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Technical Dossier

PhytoCide Black Currant PowderCode Number: M16001INCI Name: Ribes nigrum (Black Currant) Fruit Extract

abilitynaturalrowantechnologyActivitysustainabilitybenefitsEcocertleuconostoc

moistureCosmosconditionpeptideImprovingsolarchoiceantimicrobial

Technical Data SheetSpecification SheetCompositional BreakdownEfficacy Tests

a. Oxygen Radical Absorbance Capacity Assayb. Minimum Inhibition Concentration (MIC) Datac. Zone of Inhibition Datad. Challenge Test with 2.0% PhytoCide Black Currant Powder

Safety Informationa. Safety Statementb. in-vitro Dermal and Ocular Irritation Testsc. Direct Peptide Reactivity Assayd. OECD 442D TG in-vitro Skin Sensitizatione. Bacterial Reverse Mutation Testf. Phototoxicity Testg. OECD 202 Acute Daphnia Assayh. OECD 301B Ready Biogradability Assayi. Allergen Statement

Certificate of OriginMaterial Safety Data Sheet (GHS SDS)Additional Documentation

a. Manufacturing Flow Chartb. Certificate of Compliancec. ECOCERT and COSMOS

Table of Contents

PhytoCide Black Currant Powder Code Number: M16001INCI Name: Ribes nigrum (Black Currant) Fruit Extract

I.II.III.IV.

V.

VI.VII.VIII.

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Technical Data Sheet

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk involved in the use of its chemi-cal products since the conditions of use are beyond our control. Statements concerning the possible use of our products are not intended as recommendations to use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification. Freedom from patent infringement is not implied. All information is for investigative purposes only.

Active Micro Technologies, LLC - www.activemicrotechnologies.com - [email protected] Technology Drive - Lincolnton, NC 28092 - USA - Phone (704) 276-7100 - Fax (704) 276-7101

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BACKGROUND

Ribes nigrum, or black currant, is a small fruiting shrub native to Europe and northern Asia. The edible fruit is a dark purple color and typically grows 1 cm in diameter. Often used in jam, candy and juices, the popularity of black currants has recently increased due to its high concentration of antioxidants and vitamins, such as vitamin C.

Interestingly, black currants are also an excellent source of phytochemicals that exhibit antimicrobial properties. These phytochemicals can be isolated from the fruit for use as antimicrobial agents in cosmetic and personal care products. The desire for alternatives to traditional preservatives is not novel, however the notion of an antimicrobial with a secondary active that provides value-added benefits is relatively new. Active Micro Technologies developed PhytoCide Black Currant Powder, an extract rich in phytochemicals that not only inhibits microbial growth, but also have anti-inflammatory benefits.

Black currants are rich in both ellagitannins and anthocyanins which are polyphenols with anti-

inflammatory properties. Useful in an array of aqueous systems and emulsions specifically designed for

sensitive skin, PhytoCide Black Currant Powder is an effective antimicrobial that can also act

to preserve systems.

Code Number: M16001INCI Nomenclature: Ribes nigrum (Black Currant) Fruit ExtractINCI Status: ApprovedREACH Status: Fully CompliantCAS Number: 68606-81-5EINECS Number: 271-749-0Origin: Botanical: Ribes nigrum fruitProcessing: GMO FreeNo EthoxylationNo IrradiationNo SulphonationNo Ethylene Oxide treatmentNo HydrogenationAdditives: None-Preservatives: None-Antioxidants: NoneOther additives: NoneSolvents used: WaterAppearance: Free Flowing PowderSoluble/Miscible: Water Soluble atsuggested use levelsSuggested Use Levels: 1.0 - 3.0%Suggested Applications: Soothing,Conditioning, Antimicrobial

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BENEFITS

This incorporation will either reduce the level of traditional preservatives being used or help create a blend of natural antimicrobials. Additionally the anti-inflammatory properties of the black currant may help quell mild irritation associated with both sensitive and problem skin.

PhytoCide Black Currant Powder

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk involved in the use of its chemi-cal products since the conditions of use are beyond our control. Statements concerning the possible use of our products are not intended as recommendations to use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification. Freedom from patent infringement is not implied. All information is for investigative purposes only.

Active Micro Technologies, LLC - www.activemicrotechnologies.com - [email protected] Technology Drive - Lincolnton, NC 28092 - USA - Phone (704) 276-7100 - Fax (704) 276-7101

PhytoCide Black Currant Powder is stable and efficacious at temperatures up to 75°C and a pH between 3 and 8. Not only can this unique multifunctional ingredient be used to deliver the numerous benefits associated with black currants, but it can also be used as a natural antimicrobial agent in a wide variety of cosmetic and personal care applications.

Minimum Inhibitory Concentrations (MIC), using a standard growth media dilution method in addition to both bacterial and fungal cultures, were determined in order to evaluate the ability of PhytoCide Black Currant Powder to protect against microbial proliferation. The results shown in Figure 1 indicate PhytoCide Black Currant Powder can provide effective protection for cosmetic formulations.

Figure 1. MIC data for PhytoCide Black Currant Powder

Microorganism Tested MIC (%)E. coli 1.00P. aeruginosa 0.50S. aureus 1.00 C. albicans 1.00A. brasiliensis 2.00

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Table 2. Challenge Test results for 2% PhytoCide Black Currant Powder in a cream base inoculated and tested on day 7, 14, 21 and 28 and then re-inoculated and tested on day 7, 14, 21, and 28.

2.6 X 105

Day 7Day 14Day 21Day 28

S. aureusP. aeruginosaE. coli C. albicans A. brasiliensis

Inoculum Level (initial)

Inoculum (re-inoculated)

3.6 X 106 1.0 X 106 1.4 X 106 2.0 X 107 4.0 X 105

>99.999%>99.999% 99.856%>99.999%>99.999% >99.999% 99.896%>99.999%>99.999%

>99.999% >99.999%>99.999%>99.999%

99.904% 99.909%

>99.999% 99.760%99.775%99.782%99.782%

1.1 X 106 4.9 X 105 1.0 X 105 2.0 X 104

Day 7Day 14Day 21Day 28

99.900%99.961% 99.992% 91.500%99.946%99.982% >99.999% 92.000%

>99.999%>99.999% >99.999% 99.991%>99.999%>99.999% >99.999% 99.991%

96.500%97.400%99.350%99.955%

A double challenge test using 2% PhytoCide Black Currant Powder was also con ducted to evaluate the ability of the product to provide antimicro bial protection in finished personal care products. A basic O/W emulsion was used as the base. The samples were inoculated with E. coli, P. aeruginosa, S. aureus, C. albicans and A. brasilien sis and incubated for 28 days. Dur ing this period, samples were peri odically collected and tested for the presence of viable microorganisms. Following the initial 28 days of incu bation, the samples were re-inocu lated with the microbial cultures for another period of 28 days. The re sults are illustrated in Table 2.


This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration.

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107 Technology Drive Lincolnton, NC 28092 (704) 276-7100 Fax (704) 276-7101

Specification

Product Name: PhytoCide Black Currant Powder Code Number: M16001 CAS #’s: 68606-81-5 EINECS #’s: 271-749-0INCI Name: Ribes nigrum (Black Currant) Fruit Extract

Specification Parameter

Appearance

Color

Odor

pH (1% Solution in Water)

Loss on Drying (1g-1hr-1050C)

Solubility (in Water)

Citric Acid

Heavy Metals (Total)

Lead

Arsenic

Mercury

Free Flowing Powder

Light Pink with Darker Specks

Characteristic

3.0 – 6.0

8.0% Maximum

Partially Water Soluble

1.5 – 4.0%

< 20 ppm

< 10 ppm

< 3 ppm

< 1 ppm



Compositional Breakdown 107 Technology Drive Lincolnton, NC 28092

(704) 276-7100 Fax (704) 276-7101

PhytoCide Black Currant Powder Code: M16001

Compositional Breakdown:

Ingredient %

Ribes nigrum (Black Currant) Fruit Extract 100.00

• To our knowledge the above material is free of the following list of heavymetals:- Heavy Metals < 20 ppm (Max.)- Lead < 10 ppm (Max.)- Antimony < 5 ppm (Max.)- Arsenic < 3 ppm (Max.)- Mercury < 1 ppm (Max.)- Cadmium < 1 ppm (Max.)




(704) 276-7100 Fax (704) 276-7101

This is to certify that the following allergens were not detected in PhytoCide Black Currant Powder:

ALLERGENS Dir 2003 15 CEE

INCI NAME CAS NUMBER

Alpha-IsoMethyl Ionone 127-51-5

Amyl Cinnamal 122-40-7

Anise Alcohol 105-13-5

Benzyl Alcohol 100-51-69

Benzyl Benzoate 120-51-4

Benzyl Cinnamate 103-41-3

Benzyl Salicylate 118-58-1

Butylphenyl Methylpropional 80-54-6

Cinnamal 104-55-2

Cinnamyl Alcohol 104-54-1

Citral 5392-40-5

Citronellol 106-22-9

Coumarin 91-64-5

Eugenol 97-53-0

Farnesol 4602-84-0

Geraniol 106-24-1

Hexyl Cinnamal 101-86-0

Hydroxycitronellal 107-75-5

Hydroxymethylpentyl 3-Cyclohexene carboxaldehyde

31906-04-4

Isoeugenol 97-54-1

Limonene 5989-27-5

Linalool 78-70-6

Methyl 2 Octynoate 111-12-6

Evernia prunastri 90028-68-5

Evernia furfuracea 90028-67-4

Amylcinnamyl Alcohol 101-85-9




(704) 276-7100 Fax (704) 276-7101

This is to certify that PhytoCide Black Currant Powder does not contain pesticide levels exceeding the following:

EPA Pesticide Levels

INCI NAME LIMIT (mg/kg)

Alachlor 0.02

Aldrin and Dieldrin 0.05

Azinphos-methyl 1.00

Bromopropylate 3.00

Chlordane(cis and trans) 0.05

Chlorfenvinphos 0.50

Chlorpyrifos 0.20

Chlorpyrifos-methyl 0.10

Cypermethrin 1.00

DDT 1.00

Deltamethrin 0.50

Diazinon 0.50

Dichlorvos 1.00

Dithiocarbamates 2.00

Endosulfan 3.00

Endrin 0.05

Ethion 2.00

Fenitrothion 0.50

Fenvalerate 1.50

Fonofos 0.05

Heptachlor 0.05

Hexachlorobenzene 0.10

Hexachlorocyclohexane 0.30

Lindane 0.60

Malathion 1.00

Methidathion 0.20




(704) 276-7100 Fax (704) 276-7101

Parathion 0.50

Parathion-methyl 0.20

Permethrin 1.00

Phosalone 0.10

Piperonyl butoxide 3.00

Pirimiphos-methyl 4.00

Pyrethrins 3.00

Quintozene(sum of 3 items) 1.00




Oxygen Radical Absorbance Capacity (ORAC) Assay

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Tradename: PhytoCide Black Currant Powder

Code: M16001

CAS #: 68606-81-5

Test Request Form #: 59

Lot #: 25297

Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 Study Director: Erica Segura Principle Investigator: Meghan Darley

Test Performed: Oxygen Radical Absorbance Capacity (ORAC)

Introduction

Reactive oxygen species (ROS) are generated by normal cellular processes, environmental stresses, and UV irradiation. ROS are dangerous to cellular structures and functional molecules (i.e., DNA, proteins, lipids) as they act as strong oxidizing agents or free radicals. The oxygen radical absorbance capacity (ORAC) assay is a standard method used to assess antioxidant capacity of physiological fluids, foods, beverages, and natural products. The assay quantitatively measures a sample’s ability to quench free radicals that have the potential to react with and damage cellular components.

Oxygen Radical Absorbance Capacity (ORAC) assay was conducted to assess the antioxidant capacity of PhytoCide Black Currant Powder.

Assay Principle

This assay is based upon the effect of peroxyl radicals generated from the thermal decomposition of 2, 2’-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) on the signal intensity from the fluorescent probe, fluorescein,in the presence of an oxygen radical absorbing substance. The degree of change is indicative of the amount ofradical damage and the presence of antioxidants results in an inhibition in the free radical damage to thefluorescein. The antioxidant protection of the sample can be calculated by comparing it to a set of knownstandards. Trolox®, a water soluble vitamin E analog, with known antioxidant capabilities is used in this ORACassay as the standard for measuring the antioxidant capacity of unknown substances. ORAC values, expressedin µM of Trolox® equivalents (TE), are calculated using the area under the curves (AUC) of the test product,Trolox®, and the control materials. Trolox equivalency is used as the benchmark for antioxidant capacity ofmixtures since it is difficult to measure individual components.





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Materials

Synergy H1 Microplate reader (BioTek Instuments, Winooski, VT); Gen5 software (BioTek Instuments, Winooski, VT); Pipettes 75mM Potassium Phosphate (pH 7.4); Deionized H2O 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) (153mM); 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox®); Fluorescein Sodium Salt (4nM) Pre-heat (37°C) Synergy H1 Microplate reader; Prepare Trolox® standards,sample dilutions, fluorescein solution, and AAPH.

A. Equipment:

B. Buffers:C. Reagents:

D. Preparation:

E. Microtitre Plates: Corning 96 Well Black Side/Clear Bottom Microplates

Methods

Solutions of PhytoCide Black Currant Powder and Trolox® (positive control) were prepared in 75mM potassium phosphate buffer. Materials were prepared at three different concentrations/dilutions. Trolox® was used as a reference for antioxidant capacity and prepared a concentrations ranging from 12.5µM to 200µM in 75mM potassium phosphate buffer.

For the ORAC assay, 25µL of test material and Trolox® were combined with 150µL of fluorescein in 75mM potassium phosphate buffer and incubated in the Synergy HT Microplate reader at 37˚C for 30 minutes. At the end of the incubation period, 25µL of AAPH were pipetted into each well. Fluorescent measurements were then taken every 2 minutes for approximately 2 hours at an excitation wavelength of 485nm and an emissions wavelength of 520nm.

The AUC and Net AUC values of the standards and samples were determined using Gen5 2.0 Data Reduction Software using the below equations:

The standard curve was obtained by plotting the Net AUC of different Trolox® concentrations against their concentration. ORAC values of samples were then calculated automatically using the Gen5 software to interpolate the sample’s Net AUC values against the Trolox® standard curve. ORAC measurements for the test material were expressed in micro moles Trolox® equivalents (µMTE), where 1 ORAC unit is equal to 1 µMTE.





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Results

PhytoCide Black Currant Powder exhibited potent antioxidant activity at a 0.1% concentration.

Figure 1: Antioxidant capacities

Discussion

As shown in figure 1, PhytoCide Black Currant Powder exhibited potent antioxidant activity comparable to Trolox®. The antioxidant capacity of PhytoCide Black Currant Powder increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependant.

PhytoCide Black Currant Powder was designed to be conditioning, soothing, and provide antimicrobial properties. With the present study we can confirm that this unique ingredient is not only capable of providing functional benefits but it is also capable of providing potent antioxidant benefits when added to cosmetic applications.

Inhibition Activity Data




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Product Name: PhytoCide Black Currant Powder Code Number: M16001 Lot Number: 39441P Test Request Number: 1001 CAS #’s: 68606-81-5 EINECS #’s: 271-749-0INCI Name: Ribes nigrum (Black Currant) Fruit Extract

Organism (ATCC #) Minimum Inhibitory Concentration (%)

E.coli #8739

S. aureus #6538

P. aeruginosa #9027

C. albicans #10231

A. brasiliensis #16404

0.5

0.5

0.5

0.5

2.0

QA Signature Monica Beltran

Date 01-07-15

This is an Electronically Generated Document

Zone of Inhibition Test




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Product Name: PhytoCide Black Currant Powder Code Number: M16001 Lot Number: 40785P Test Request Number: 1086 CAS #’s: 68606-81-5 EINECS #’s: 271-749-0INCI Name: Ribes nigrum (Black Currant) Fruit Extract

Organism (ATCC #) Zone of Inhibition (mm)

E.coli #8379

S. aureus #6538

P. aeruginosa #9027

C. albicans #10231

A. brasiliensis #16404

8.0

8.0

8.0

8.3

8.0

QA Signature Monica Beltran

Date 03-09-2015

This is an Electronically Generated Document


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Challenge Test with 2.0% PhytoCide Black Currant Powder

Antimicrobial Efficacy Test PCPC Section 20

Method 3 Determination of Preservation Adequacy of Water- Miscible Personal Care Products

Test Product


Purpose

This study was initiated to determine the efficacy of a cosmetic ingredient with antimicrobial properties in a cream formulation against bioburden as a function of time.

Study Dates

The study was started on March 27th, 2014 and was completed on May 30th, 2014.

Test Organisms

1. Escherichia coli: ATCC #8739 2. Pseudomonas aeruginosa: ATCC #90273. Staphylococcus aureus: ATCC #6538 4. Aspergillus niger: ATCC #16404 5. Candida albicans: ATCC #10231

Neutralization:

Verification of neutralization of the antimicrobial properties of the product was demonstrated prior to performing the test for microbial content by inoculating the product dilution with a low level of challenge microorganisms (100 CFU) and verifying recovery of this viable inoculum. This provides evidence that the antimicrobial has been neutralized and there are no false positive results during the Challenge Test.

Test Method

Fifty grams of Generic Cream with 2% PhytoCide Black Currant Powder was weighed into five individual containers. Each container was inoculated with one of the five test organisms. The inoculum concentration for each organism was standardized using the 0.5 McFarland turbidity standard and further diluted to yield approximately 106 to 108 microorganisms/ml. The amount of each inoculum added to each sample was no more than 1% of the product weight, as to not alter the product composition.


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The inoculated samples were evaluated 7, 14, 21, and 28 days after the initial inoculation to determine quantitatively the number of viable microorganisms remaining. On the 28th day of testing the samples were re-inoculated and evaluated 7, 14, 21, and 28 days after the second exposure to determine the number of viable microorganisms. The table below represents the percent reduction of viable organisms after being introduced into the test formulation.

Table 1. Challenge Test results for Generic Cream with 2% PhytoCide Black Currant Powder inoculated on Day 7, 14, 21 and 28 then re-inoculated and tested on Day 7, 14, 21 and 28.

* The days listed in the first column refer to the inoculum/plating day. Bacteria results are read 2 days after plating day, and mold and yeastresults are read 5 days after plating day.

Results & Discussion

The results obtained from the Neutralization Test of each product using Dey/Engley (D/E) broth, indicate that the neutralization steps conducted prior to performing the Challenge Test are indeed effective for avoiding false positive Challenge Test results.

Organisms

Inoculum (initial) CFU/ml

E. coli P. aeruginosa S. aureus C. albicans A. brasiliensis

1.0 x 106 1.4 x 106 3.6 x 106 2.0 x 107 4.0 x 105

Day 7 >99.999% >99.999% >99.999% 99.856% 99.760%

Day 14 >99.999% >99.999% >99.999% 99.896% 99.775%

Day 21 >99.999% >99.999% >99.999% 99.904% 99.782%

Day 28 >99.999% >99.999% >99.999% 99.909% 99.782%

Inoculum (re-inoculated)

CFU/ml 1.1 x 106 4.9 x 105 2.6 x 105 1.0 x 105 2.0 x 104

Day 7 99.961% 99.992% 99.900% 91.500% >99.999%

Day 14 99.982% >99.999% 99.946% 92.000% 97.400%

Day 21 >99.999% >99.999% >99.999% 99.991% 99.350%

Day 28 >99.999% >99.999% >99.999% 99.991% 99.955%


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The results of this Challenge Test demonstrate the effectiveness of the preservation system used in Generic Cream with 2% PhytoCide Black Currant Powder. The recommendations stated in Section 13, Determination of Preservative Adequacy in Cosmetic Formulations, in the PCPC Microbiology Guidelines are as follows:

Bacteria – There should be at least a 99.9% (3 log) reduction of vegetative bacteria within 7 days following each challenge and no increase for the duration of the test period.

Yeasts and Molds – There should be at least a 90% (1 log) reduction of yeasts and molds within 7 days following each challenge and no increase for the duration of the test period.

The Gram positive and Gram negative bacteria as well as the yeast were reduced by greater than 99.9% within 7 days of each challenge. The mold was reduced by greater than 90% within 7 days of each challenge. By the end of each 28-day test period Gram positive and Gram negative bacteria as well as the yeast were reduced by 99.999% or greater. Mold was reduced by 99.0% or greater.


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Antimicrobial Efficacy (Challenge) Testing

The intent of performing an Antimicrobial Efficacy or Challenge test is to evaluate whether an antimicrobial agent or preservation system in a given cosmetic formulation has the ability to prevent the growth of test microorganisms. The test methodology employed by Active Micro Technologies (AMT) is based on the methods published in the CTFA Microbiology Guidelines. AMT’s goal is to assist our customers by providing a screening test of a product formulation that is approaching finalization. It is expected that the formulation(s) submitted for Challenge testing contain AMT antimicrobials and have already passed the customer’s internal stability tests. It is also anticipated that formal challenge testing of the final formulation will subsequently be performed by the customer at an outside lab of their choosing.

The information contained in this report is provided by Active Micro Technologies after the exercise of all reasonable care and skill in its compilation, preparation, and issue. It is provided without liability regarding its subsequent application and use. This type of screening test will be conducted only for validation of the efficacy of the antimicrobial agent or preservative system in the specific formulation tested. It does not address the suitability of the overall formula, nor does it address the regulatory status of any component therein. This testing does not account for the possibility of environmental microorganisms and cannot be relied upon as sufficient to justify commercialization of the product tested. By submitting samples for testing, the customer acknowledges that they will not hold Active Micro Technologies responsible for products launched based solely on the support of these studies.


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Safety Statement


Product Name: PhytoCide Black Currant Powder

Product Code: M16001

INCI Name: Ribes nigrum (Black Currant) Fruit Extract

INCI Status: Approved

PhytoCide Black Currant Powder is manufactured by aqueous extraction of black currant fruit under controlled conditions. The extract is then filtered.

The FDA (Food and Drug Administration) states in sections 201 and 409 of the Federal Food, Drug and Cosmetic Act that “any substance that is intentionally added to food is a food additive, that is subject to review and approval by FDA, unless the substance is generally recognized, among qualified experts, as having been adequately shown to be safe under conditions of its use or unless the use of the substance is otherwise excluded for the definition of a food additive.”1

Ribes nigrum is widely used in culinary preparations, and is a common ingredient in desserts and beverages especially. The fruit is also utilized in nutritional supplements due to its phytochemical content. Therefore, the Federal Food, Drug and Cosmetic Act classifies materials such as PhytoCide Black Currant Powder as Generally Recognized as Safe (GRAS). This knowledge combined with the toxicity data provided allows us to support the safety of PhytoCide Black Currant Powder in cosmetic applications at the recommended use level of 1 - 3%.

Due to the restriction placed on the animal testing of cosmetic raw materials, and our internal non-animal testing policy Active Micro Technologies, LLC does not test for NOAEL.

1. Federal Food, Drug and Cosmetic Act. U.S Food and Drug Administration. www.fda.gov.




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Dermal and Ocular Irritation Test

Tradename: Phytocide Black Currant Powder

Code: M16001

CAS #: 84603-58-7


Lot #: SN120626-7


Test Performed: In Vitro EpiDerm™ Dermal Irritation Test (EPI-200-SIT) EpiOcular™ Eye Irritation Test (OCL-200-EIT)

SUMMARY

In vitro dermal and ocular irritation studies were conducted to evaluate whether Phytocide Black Currant Powder would induce dermal or ocular irritation in the EpiDerm™ and EpiOcular™ model assays.

The product was tested according to the manufacture’s protocol. The test article solution was found to be a non-irritant. Reconstructed human epidermis and cornea epithelial model were incubated in growth media overnight to allow for tissue equilibration after shipping from MatTek Corporation, Ashland, MA. Test substances were applied to the tissue inserts and incubated for 60 minutes for liquid and solid substances in the EpiDerm™ assay and 30 minutes for liquid substances and 90 minutes for solid substances in the EpiOcular™ assay at 37°C, 5% CO2, and 95% relative humidity (RH). Tissue inserts were thoroughly washed and transferred to fresh plates with growth media. After post substance dosing incubation is complete, the cell viability test begins. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl)], present in the cell mitochondria, into blue formazan salt that is measured after extraction from the tissue. The irritation potential of the test chemical is dictated by the reduction in tissue viability of exposed tissues compared to the negative control.

Under the conditions of this assay, the test article was considered to be non-irritating. The negative and positive controls performed as anticipated.

I. IntroductionA. PurposeIn vitro dermal and ocular irritation studies were conducted to evaluate whether a test article would induce dermal orocular irritation in the EpiDerm™ and EpiOcular™ model assays. MatTek Corporation’s reconstructed humanepidermal and human ocular models are becoming a standard in determining the irritancy potential of testsubstances. They are able to discriminate between irritants and non-irritants. The EpiDerm™ assay has accuracy forthe prediction of UN GHS R38 skin irritating and no-label (non-skin irritating) test substances. The EpiOcular™ assaycan differentiate chemicals that have been classified as R36 or R41 from the EU classifications based on DangerousSubstances Directive (DSD) or between the UN GHS Cat 1 and Cat 2 classifications.




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II. MaterialsA. Incubation Conditions: 37°C at 5% CO2 and 95% relative humidityB. Equipment: Forma humidified incubator, ESCO biosafety laminar flow hood, Synergy HT

Microplate reader; Pipettes C. Media/Buffers: DMEM based medium; DPBS; sterile deionized H2O D. Preparation: Pre-incubate (37°C) tissue inserts in assay medium; Place assay medium and MTT

diluent at 4°C, MTT concentrate at -20°C, and record lot numbers of kit components E. Tissue Culture Plates: Falcon flat bottom 96-well, 24-well, 12-well, and 6-well tissue culture plates F. Reagents: MTT (1.0mg/mL); Extraction Solution (Isopropanol); SDS (5%); Methyl Acetate G. Other: Nylon Mesh Circles (EPI-MESH); Cotton tip swabs; 1mL tuberculin syringes; Ted Pella

micro-spatula; 220mL specimen containers; sterile disposable pipette tips; Parafilm

III. Test AssayA. Test SystemThe reconstructed human epidermal model, EpiDerm™, and cornea epithelial model, EpiOcular™, consist of normalhuman-derived epidermal keratinocytes which have been cultured to form a multilayer, highly differentiated model ofthe human epidermis and cornea epithelium. These models consist of organized basal, spinous, and granular layers,and the EpiDerm™ systems also contains a multilayer stratum corneum containing intercellular lamellar lipid layersthat the EpiOcular™ system is lacking. Both the EpiDerm™ and EpiOcular™ tissues are cultured on specially preparedcell culture inserts.

B. Negative ControlSterile DPBS and sterile deionized water are used as negative controls for the EpiDerm™ and EpiOcular™ assays,respectfully.

C. Positive ControlKnown dermal and eye irritants, 5% SDS solution and Methyl Acetate, were used as positive controls for theEpiDerm™ and EpiOcular™ assays, respectfully.

D. Data Interpretation Procedurea. EpiDerm™An irritant is predicted if the mean relative tissue viability of the 3 tissues exposed to the test substance isreduced by 50% of the mean viability of the negative controls and a non-irritant’s viability is ˃ 50%.b. EpiOcular™An irritant is predicted if the mean relative tissue viability of the 2 tissues exposed to the test substance isreduced by 60% of the mean viability of the negative controls and a non-irritant’s viability is ˃ 40%.

IV. MethodA. Tissue ConditioningUpon MatTek kit arrival at Active Concepts, LLC the tissue inserts are removed from their shipping medium andtransferred into fresh media and tissue culture plates and incubated at 37°C at 5% CO2 and 95% relative humidity for60 minutes. After those 60 minutes the inserts are transferred into fresh media and tissue culture plates andincubated at 37°C at 5% CO2 and 95% relative humidity for an additional 18 to 21 hours.




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B. Test Substance Exposurea. EpiDerm™30µL (liquid) or 25mg (solid) of the undiluted test substance is applied to 3 tissue inserts and allowed toincubate for 60 minutes in a humidified incubator (37°C, 5% CO2, 95% RH).b. EpiOcular™Each tissue is dosed with 20µL DPBS prior to test substance dosing. 50µL (liquid) or 50mg (solid) of theundiluted test substance is applied to 2 tissue inserts and allowed to incubate for 90 minutes in a humidifiedincubator (37°C, 5% CO2, 95% RH).

C. Tissue Washing and Post Incubationa. EpiDerm™All tissue inserts are washed with DPBS, dried with cotton tipped swab, and transferred to fresh media andculture plates. After 24 hours the inserts are again transferred into fresh media and culture plates for anadditional 18 to 20 hours.b. EpiOcular™Tissue inserts are washed with DPBS and immediately transferred into 5mL of assay medium for 12 to 14minutes. After this soak the inserts are transferred into fresh media and tissue culture plates for 120 minutesfor liquid substances and 18 hours for solid substances.

D. MTT AssayTissue inserts are transferred into 300µL MTT media in pre-filled plates and incubated for 3 hours at 37°C, 5% CO2,and 95% RH. Inserts are then removed from the MTT medium and placed in 2mL of the extraction solution. Theplate is sealed and incubated at room temperature in the dark for 24 hours. After extraction is complete the tissueinserts are pierced with forceps and 2 x 200µL aliquots of the blue formazan solution is transferred into a 96 wellplate for Optical Density reading. The spectrophotometer reads the 96-well plate using a wavelength of 570 nm.

V. Acceptance CriterionA. Negative ControlThe results of this assay are acceptable if the mean negative control Optical Density (OD570) is ≥ 1.0 and ≤ 2.5(EpiDerm™) or ≥ 1.0 and ≤ 2.3 (EpiOcular™).

B. Positive Controla. EpiDerm™The assay meets the acceptance criterion if the mean viability of positive control tissues expressed as a % ofthe negative control is ≤ 20%.b. EpiOcular™The assay meets the acceptance criterion if the mean viability of positive control tissues is < 60% of controlviability.

C. Standard DeviationSince each irritancy potential is predicted from the mean viability of 3 tissues for EpiDerm™ and 2 tissues forEpiOcular™, the variability of the replicates should be < 18% for EpiDerm™ and < 20% EpiOcular™.

VI. ResultsA. Tissue CharacteristicsThe tissue inserts included in the MatTek EpiDerm™ and EpiOcular™ assay kits were in good condition, intact, andviable.




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B. Tissue Viability AssayThe results are summarized in Figures 1 and 2. In no case was the tissue viability ≤ 50% for EpiDerm™ or ≤ 60%for EpiOcular™ in the presence of the test substance. The negative control mean exhibited acceptable relative tissueviability while the positive control exhibited substantial loss of tissue viability and cell death.

C. Test ValidityThe data obtained from this study met criteria for a valid assay.

VII. ConclusionUnder the conditions of this assay, the test article substance was considered to be non-irritating. The negative and positive controls performed as anticipated.

Figure 1: EpiDerm tissue viability

Figure 2: EpiOcular tissue viability


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OECD TG 442C: In Chemico Skin Sensitization


Page 1 of 4


Code: M16001

CAS #: 68606-81-5


Lot #: 41416P

Sponsor: Active Micro Technologies, LLC; 107 Technology Drive Lincolnton, NC 28092 Study Director: Erica Segura Principle Investigator: Meghan Darley

Test Performed: OECD TG 442C: In Chemico Skin Sensitization

Direct Peptide Reactivity Assay (DPRA)

Introduction

A skin sensitizer is a substance that will lead to an allergic response following skin contact1. Haptenation is the covalent binding of a hapten, or low-molecular weight substance or chemical, to proteins in the skin. This is considered the prominent mechanism which defines a chemical as a sensitizer. Haptenation is described as a "molecular initiating event" in the OECD Adverse Outcome Pathway (AOP) for skin sensitization which summarizes the key events known to be involved in chemically-induced allergic contact dermatitis2. The direct peptide reactivity assay (DPRA) is designed to mimic the covalent binding of electrophilic chemicals to nucleophilic centers in skin proteins by quantifying the reactivity of chemicals towards the model synthetic peptides containing cysteine and lysine. The DPRA is able to distinguish sensitizers from non-sensitizer with 82% accuracy (sensitivity of 76%; specificity of 92%)3.

This assay was conducted to determine skin sensitization hazard of PhytoCide Black Currant Powder in accordance with European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and OECD Test Guideline 442C.

Assay Principle

The DPRA is an in chemico method which addresses peptide reactivity by measuring depletion of synthetic heptapeptides containing either cysteine or lysine following 24 hours incubation with the test substance. The peptide is a custom material containing phenylalanine to aid in detection. Depletion of the peptide in the reaction mixture is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

1. United Nations Economic Commission (UNECE) (2013) Global Harmonized System of Classification and Labelling of Chemicals (GHS) 5th Revised Edition 2. OECD (2012). The Adverse Outcome Pathway for Skin Sensitization Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 1683. EC EURL ECVAM (2012) Direct peptide reactivity assay (DPRA) validation study report; pp 1 -74.


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Page 2 of 4

Materials

A. Equipment: HPLC-UV (Waters Breeze - Waters 2998 Photodiode Array Detector); Pipettes; Analytical balance

B. HPLC/Guard Columns: Agilent Zorbax SB-C18 2.1mm x 100mm x 3.5µm; Phenomenex Security Guard C18 4mm x 2mm

C. Chemicals: Trifluoroacetic acid; Ammonium acetate; Ammonium hydroxide; Acetonitrile; Cysteine peptide (Ac-RFAACAA-COOH); Lysine peptide (Ac-RFAAKAA-COOH); Cinnamic aldehyde

D. Reagents/Buffers: Sodium phosphate buffer (100mM); Ammonium acetate buffer (100mM)

E. Other: Sterile disposable pipette tips

Methods

Solution Preparation:

• 0.667mM Cysteine Peptide in 100mM Phosphate Buffer (pH 7.5)• 0.667mM Lysine Peptide in 100mM Ammonium Acetate Buffer (pH 10.2)• 100mM Cinnamic Aldehyde in Acetonitrile• 100mM PhytoCide Black Currant Powder in Acetonitrile

Reference Controls:

• Reference Control A: For calibration curve accuracy• Reference Control B: For peptide stability over analysis time of experiment• Reference Control C: For verification that the solvent does not impact percent peptide depletion

Sample, Reference Control, and Co-Elution Control Preparation:

• Once these solutions have been made they should be incubated at room temperature, protected from light,for 24±2 hours before running HPLC analysis.

• Each chemical should be analyzed in triplicate.

1:10 Ratio, Cysteine Peptide 0.5mM Peptide, 5mM Test Chemical

1:50 Ratio, Lysine Peptide 0.5mM Peptide, 25mM Test Chemical

• 750µL Cysteine Peptide Solution(or 100mM Phosphate Buffer, pH 7.5, for Co-ElutionControls)

• 200µL Acetonitrile• 50µL Test Chemical Solution

(or Acetonitrile for Reference Controls)

• 750µL Lysine Peptide Solution(or 100mM Ammonium Acetate Buffer, pH 10.2,for Co-Elution Controls)

• 250µL Test Chemical Solution(or Acetonitrile for Reference Controls)


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Calibration Curve:

• Standards are prepared in a solution of 20% Acetonitrile:Buffero For the Cysteine peptide using the phosphate buffer, pH 7.5o For the Lysine peptide using the ammonium acetate buffer, pH 10.2

Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 mM Peptide 0.534 0.267 0.1335 0.0667 0.0334 0.0167 0.000

HPLC Analysis:

• HPLC-UV system should be equilibrated at 30°C with 50% Mobile Phase A (0.1% (v/v) trifluoroacetic acidin water) and 50% Mobile Phase B (0.085% (v/v) trifluoroacetic acid in acetonitrile) for 2 hours

• Absorbance is measured at 220nm• Flow Conditions:

Time Flow %A %B 0 minutes 0.35 mL/min 90 10 10 minutes 0.35 mL/min 75 25 11 minutes 0.35 mL/min 10 90 13 minutes 0.35 mL/min 10 90 13.5 minutes 0.35 mL/min 90 10 20 minutes End Run

Data and Reporting

Acceptance Criteria:

1. The following criteria must be met for a run to be considered valid:a. Standard calibration curve should have an r2 > 0.99.b. Mean percent peptide depletion values of three replicates for the positive control cinnamic

aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and69% for the lysine peptide and the maximum standard deviation should be <14.9 for the percentcysteine depletion and <11.6 for the percent lysine depletion.

c. Mean peptide concentration of reference controls A should be 0.50±0.05mM and the coefficient ofvariable of the peptide peak areas for reference B and C in acetonitrile should be <15.0%.

2. The following criteria must be met for a test chemical’s results to be considered valid:a. Maximum standard deviation should be <14.9 for percent cysteine depletion and <11.6 for percent

lysine depletion.b. Mean peptide concentration of the three reference control C should be 0.50±0.05mM.


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Page 4 of 4

Prediction Model:

Cysteine 1:10/Lysine 1:50 Prediction Model Mean of Cysteine and Lysine % Depletion Reactivity Class Prediction

0% < Mean % Depletion < 6.38% Minimal Reactivity Non-sensitizer 6.38% < Mean % Depletion < 22.62% Low Reactivity Sensitizer 22.62% < Mean % Depletion < 42.47% Moderate Reactivity Sensitizer 42.47% < Mean % Depletion < 100% High Reactivity Sensitizer

If co-elution occurs with the lysine peptide, than the cysteine 1:10 prediction model can be used:

Cysteine 1:10 Prediction Model Mean of Cysteine and Lysine % Depletion Reactivity Class Prediction

0% < Cys % Depletion < 13.89% Minimal Reactivity Non-sensitizer 13.89% < Cys % Depletion < 23.09% Low Reactivity Sensitizer 23.09% < Cys % Depletion < 98.24% Moderate Reactivity Sensitizer 98.24% < Cys % Depletion < 100% High Reactivity Sensitizer

Results and Discussion

The data obtained from this study met criteria for a valid assay and the controls performed as anticipated.

Percent peptide depletion is determined by the following equation:

𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐷𝐷𝑃𝑃𝑃𝑃𝐷𝐷𝑃𝑃𝑃𝑃𝑃𝑃𝐷𝐷𝑃𝑃 = �1 − � 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐴𝐴𝐴𝐴𝑃𝑃𝑃𝑃 𝑃𝑃𝑖𝑖 𝑅𝑅𝑃𝑃𝑃𝑃𝑅𝑅𝑃𝑃𝑅𝑅𝑃𝑃𝑃𝑃𝑃𝑃 𝐼𝐼𝑖𝑖𝐼𝐼𝑃𝑃𝑅𝑅𝑃𝑃𝑃𝑃𝐼𝐼𝑖𝑖𝑀𝑀𝑃𝑃𝑃𝑃𝑖𝑖 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐴𝐴𝐴𝐴𝑃𝑃𝑃𝑃 𝑃𝑃𝑖𝑖 𝑅𝑅𝑃𝑃𝑅𝑅𝑃𝑃𝐴𝐴𝑃𝑃𝑖𝑖𝑅𝑅𝑃𝑃 𝐶𝐶𝐼𝐼𝑖𝑖𝑃𝑃𝐴𝐴𝐼𝐼𝑅𝑅𝐶𝐶 𝐶𝐶

�� × 100

Based on HPLC-UV analysis of PhytoCide Black Currant Powder (code M16001) we can determine that this product is not a sensitizer and will not cause allergic contact dermatitis. The Mean Percent Depletion of Cysteine and Lysine was 3.39% causing minimal reactivity in the assay giving us the prediction of a non-sensitizer.


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OECD TG 442D: In Vitro Skin Sensitization


Page 1 of 3


Code: M16001

CAS #: 68606-81-5


Lot #: 41416P

Sponsor: Active Micro Technologies, LLC; 107 Technology Drive Lincolnton, NC 28092 Study Director: Erica Segura Principle Investigator: Meghan Darley

Test Performed: OECD TG 442D: In Vitro Skin Sensitization

ARE-Nrf2 Luciferase Test Method

Introduction

Skin sensitization refers to an allergic response following skin contact with the tested chemical, as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals1. Substances are classified as skin sensitizers if there is evidence in humans that the substance can lead to sensitization by skin contact or positive results from appropriate tests, both in vivo and in vitro. Utilization of the KeratinoSens™ cell line allows for valid in vitro testing for skin sensitization.

This assay was conducted to determine skin sensitization potential of PhytoCide Black Currant Powder in accordance with the UN GHS.

Assay Principle

The ARE-Nrf2 luciferase test method addresses the induction of genes that are regulated by antioxidant response elements (ARE) by skin sensitizers. The Keap1-Nrf2-ARE pathways have been shown to be major regulator of cytoprotective responses to oxidative stress or electrophilic compounds. These pathways are also known to be involved in the cellular processes in skin sensitization. Small electrophilic substances such as skin sensitizers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes.

The skin sensitization assay utilizes the KeratinoSens™ method which uses an immortalized adherent human keratinocyte cell line (HaCaT cell line) that has been transfected with a selectable plasmid to quantify luciferase gene induction as a measure of activation of Keap1-Nrf2-antioxidant/electrophile response element (ARE). This test method has been validated by independent peer review by the EURL-ECVAM. The addition of a luciferin containing reagent to the cells will react with the luciferase produced in the cell resulting in luminescence which can be quantified with a luminometer.

1. United Nations (UN) (2013). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Fifth revised edition, UN New York and Geneva, 2013

This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied.This information is offered solely for your investigation, verification, and consideration.

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Page 2 of 3

Materials

A. Incubation Conditions: 37°C at 5% CO2 and 95% relative humidity (RH)B. Equipment: Humidified incubator; Biosafety laminar flow hood; Microplate Reader;

Pipettes C. Cell Line: KeratinoSens™ by Givaudan Schweiz AG D. Media/Buffers: Dulbecco's Modified Eagle Medium (DMEM); Fetal Bovine Serum

(FBS); Phosphate Buffered Saline (PBS); Geneticin E. Culture Plate: Flat bottom 96-well tissue culture treated plates F. Reagents: Dimethyl Sulfoxide (DMSO); Cinnamic Aldehyde; ONE-Glo Reagent;

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); sodium lauryl sulfate (SLS)

G. Other: Sterile disposable pipette tips; wash bottles

Methods

KeratinoSens™ were into seeded four 96-well tissue culture plates and allowed to grow to 80 – 90% confluency in DMEM containing 10% FBS and 500µg/mL G418 geneticin. Twelve test concentrations of PhytoCide Black Currant Powder were prepared in DMSO with a concentration range from 0.098 – 200µM. These 12 concentrations were assayed in triplicate in 2 independently performed experiments. The positive control was cinnamic aldehyde for which a series of 5 concentrations prepared in DMSO had final test concentrations of 4 – 64µM. The negative control was a 1% test concentration of DMSO.

24 hour post KeratinoSens™ seeding, the culture media was removed and replaced with fresh media containing 10% FBS without G418 geneticin. 50 µL of the above described test concentrations was added to the appropriate wells. The treated plates were then incubated for 48 hours at 37°C in the presence of 5% CO2 and 95% relative humidity.After treatment incubation was complete the media was removed and the wells were washed with PBS 3 times.

One of the four plates was used for a cytotoxicity endpoint, where MTT was added to the wells and incubated for 4 hours at 37°C in the presence of 5% CO2. SLS was then added to the wells and incubated overnight at roomtemperature. A spectrometer measured the absorbance at 570 nm. The absorbance values (optical density) were then used to determine the viability of each well by comparing the optical density of each test material treated well to that of the solvent control wells to determine the IC50 and IC30 values.

The remaining 3 plates were used in the luciferase induction endpoint of the assay. 100 µL of Promega’s ONE-Glo Reagent was added to 100 µL of fresh media containing 10% FBS without geneticin. Cells were incubated for 5 minutes to induce cell lysis and release luciferin into the media. Plates were read with a luminometer and EC1.5 and maximum response (Imax) values were obtained.

Data and Reporting

Acceptance Criteria:

1. Gene induction obtained with the positive control, cinnamic aldehyde, should be statistically significantabove the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM).

2. The EC1.5 value should be within two standard deviations of the historical mean and the average inductionin the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8.

3. The average coefficient of variability of the luminescence reading for the negative (solvent) control DMSOshould be below 20% in each experiment.


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A KeratinoSens™ prediction is considered positive if the following conditions are met:

1. The Imax is higher than 1.5-fold and statistically significantly higher as compared to the solvent (negative)control

2. The cellular viability is higher than 70% at the lowest concentration with a gene induction above 1.5 fold(i.e., at the EC1.5 determining concentration)

3. The EC1.5 value is less than 1000 µM (or < 200 µg/ml for test chemicals with no defined MW)4. There is an apparent overall dose-response for luciferase induction

Results

Compound Classification EC1.5 (µM) IC50 Imax

Cinnamic aldehyde Sensitizer 19 289.19 µM 31.6 DMSO Non-Sensitizer No Induction 243.24 µM 1.2

PhytoCide Black Currant Powder Non-Sensitizer No Induction > 1000 µM 0.5

Table 1: Overview of KeratinoSens™ Assay Results

Figure 1: Fold Induction of Luciferase

Discussion

As shown in the results, PhytoCide Black Currant Powder (code M16001) was not predicted to be a skin sensitizer based on the KeratinoSens™ ARE-Nrf2 Luciferase Test Method as there was not a significant increase in luciferase expression. It can be concluded that PhytoCide Black Currant Powder can be safely used in cosmetics and personal care products at typical use levels.

0

1

2

3

4

5

6

50 µm 90 µM 125 µM 50 µM 90 µM 125 µM 50 µM 90 µM 125 µM

Cinnamic aldehyde DMSO PhytoCide Black CurrantPowder

Fold

Indu

ctio

n of

Luc

ifera

se A

ctiv

ity

KeratinoSens™ AssayM16001 PhytoCide Black Currant Powder




Page 1 of 8

Bacterial Reverse Mutation Test

Test Article: PhytoCide Black Currant Powder Sponsor: Code Number: M16001 Active Micro Technologies, LLC CAS #: 999999-99-4 107 Technology Drive

Lincolnton, NC 28092

Study Director: Erica Segura Principle Investigator: Monica Beltran

Test Performed: Reference: Genotoxicity: Bacterial Reverse Mutation Test OECD471/ISO10993.Part 3

Test Request Number: 1039

SUMMARY

A Salmonella typhimurium/Escherichia coli reverse mutation standard plate incorporation study described by Ames et al. (1975) was conducted to evaluate whether a test article in solution PhytoCide Black Currant Powder would cause mutagenic changes in the average number of reveratants for histidine-dependent Salmonella typhimurium strains TA98, TA100, TA1537, TA1535 and tryptophan-dependent Escherichia coli strain WP2uvrA in the presence and absence of Aroclor-induced rat liver S9. This study was conducted to satisfy, in part, the Genotoxicity requirement of the International Organization for Standardization: Biological Evaluation of Medical Devices, Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity.

The stock test article was tested at eight doses levels along with appropriate vehicle control and positive controls with overnight cultures of tester strains. The test article in solution was found to be noninhibitory to growth of tester strain TA98, TA100, TA1537, TA1535 and WP2uvrA after Sport Inhibition Screen.

Separate tubes containing 2 ml of molten top agar at 450C supplemented with histidine-biotin solution for the Salmonella typhimurium strains and supplemented with tryptophan for Escherichia coli strain were inoculated with 100 µl of tester strains, 100 µl of vehicle or test article dilution were added and 500 µl aliquot of S9 homogenate, simulating metabolic activation, was added when necessary. After vortexing, the mixture was poured across the Minimal Glucose Agar (GMA) plates. Parallel testing was also conducted with positive control correspond to each strain, replacing the test article aliquot with 50µl aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 hours at 370C. The mean numbers of revertants of the test plates were compared to the mean number of revertants of the negative control plates for each of the strains tested. The means obtained for the positive controls were used as points of reference.

Under the conditions of this assay, the test article in solution was considered to be Non-Mutagenic to Salmonella typhimurium tester strains TA98, TA100, TA1537, TA1535 and Escherichia coli tester strain WP2uvrA. The negative and positive controls performed as anticipated. The results of this study should be evaluated in conjunction with other required tests as listed in ISO 100993, Part 3: Tests for Genotoxicity, Carcinogenicity, and Reproductive Toxicology.

I. Introduction

A. PurposeA Salmonella typhimurium/Escherichia coli reverse mutation standard plate incorporation study was conducted toevaluate whether a test article solution would cause mutagenic changes in the average number of revertants forSalmonella typhimurium tester strains TA98, TA100, TA1537, TA1535 and Escherichia coli WP2uvrA in the presenceand absences of the S9 metabolic activation. Bacterial reverse mutation tests have been widely used as rapidscreening procedures for the determination of mutagenic and potential carcinogenic hazards.




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II. Materials

A. Storage Conditions: Room temperature (23-25C).B. Vehicle: Sterile DI Water. C. Preparation: Eight different doses level were prepared immediately before use with sterile DI water. D. Solubility/Stability: 100% Soluble and Stable.E. Toxicity: No significant inhibition was observed.

III. Test System

A. Test SystemEach Salmonella typhimurium and Escherichia coli tester strain contains a specific deep rough mutation (rfa), thedeletion of uvrB gene and the deletion in the uvrA gene that increase their ability to detect mutagens, respectively.These genetically altered Salmonella typhimurium strains (TA98, TA100, TA1537 and TA1535) and Escherichia colistrain (WP2uvrA) cannot grow in the absence of histidine and tryptophan, respectively. When placed in a histidine-tryptophan free medium, only those cells which mutate spontaneously back to their wild type states are able to formcolonies. The spontaneous mutation rate (or reversion rate) for any one strain is relatively constant, but if a mutagenis added to the test system, the mutation rate is significantly increased.

Tester strain Mutations/Genotypic Relevance TA98 hisD3052, Dgal chlD bio uvrB rfa pKM101 TA100 hisG46, Dgal chlD BIO uvrB rfa pKM101 TA1537 hisC3076, rfa, Dgal chlD bio uvrB TA 1535 hisG46, Dgal chlD bio uvrB rfa WP2uvrA trpE, uvrA

rfa = causes partial loss of the lip polysaccharide wall which increases permeability of the cell to large molecules.

uvrB = deficient DNA excision-repair system (i.e., ultraviolet sensitivity) pKM101 = plasmid confers ampicillin resistance (R-factor) and enhances

sensitivity to mutagens. uvrA = All possible transitions and transversions, small deletions.

B. Metabolic ActivationAroclor induced rat liver (S9) homogenate was used as metabolic activation. The S9 homogenate is prepared frommale Sprague Dawley rats. Material is supplied by MOLTOX, Molecular Toxicology, Inc.

C. Preparation of Tester strainsCultures of Salmonella typhimurium TA98, TA100,TA1537, TA1535 and Escherichia coli WP2uvrA were inoculated toindividual flasks containing Oxoid broth No.2. The inoculated broth cultures were incubated at 37°C in an incubatorshaker operating at 140-150 rpm for 12-16 hours.

D. Negative ControlSterile DI water (vehicle without test material) was tested with each tester strain to determine the spontaneousreversion rate. Each strain was tested with and without S9 activation. These data represented a base rate to whichthe number of reveratants colonies that developed in each test plate were compared to determine whether the testmaterial had significant mutagenic properties.

E. Positive ControlA known mutagen for each strain was used as a positive control to demonstrate that tester strains were sensitive tomutation to the wild type state. The positive controls are tested with and without the presence of S9 homogenate.




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F. Titer of the Strain Cultures:Fresh cultures of bacteria were grown up to the late exponential or early stationary phase of growth; to confirm this,serial dilutions from each strain were conducted, indicating that the initial population was in the range of 1 to 2x109/ml.

IV. Method

A. Standard Plate Incorporation Assay:Separate tubes containing 2 ml of molten top agar supplemented with histidine-biotin solution for the Salmonellatyphimurium and tryptophan for Escherichia coli were inoculated with 100 µl of culture for each strain and 100 µl oftesting solution or vehicle without test material. A 500 µl aliquot of S9 homogenate, simulating metabolic activation,was added when necessary. The mixture was poured across Minimal Glucose Agar plates labeled with strain numberand S9 activation (+/-). When plating the positive controls, the test article aliquot was replaced by 50µl aliquot ofappropriate positive control. The test was conducted per duplicate. The plates were incubated for 37°C for 2 days.Following the incubation period, the revertant colonies on each plate were recorded. The mean number of reveratntswas determined. The mean numbers of revertants of the test plates were compared to the mean number of reverantsof the negative control of each strain used.

V. Evaluation

For the test solution to be evaluated as a test failure or “potential mutagen” there must have been a 2-fold or greater increase in the number of mean revertants over the means obtained from the negative control for any or all strains. Each positive control mean must have exhibited at least a 3-fold increase over the respective negative control mean of the Salmonella tester strain used.

VI. Results and Discussion

A. Solubility:Water was used as a solvent. Solutions from the test article were made from 0.015 to 50mg/ml.

B. Dose levels tested:The maximum dose tested was 5000 µg per plate. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and5000 µg per plate.

C. Titer (Organisms/ml):5 x 108 UFC/ml plate count indicates that the initial population was in the range of 1 to 2 x 109 UFC/ml.

C. Standard Plate Incorporation AssayIn no case was there a 2-fold or greater increase in the mean number of revertant testing strains TA98, TA100,TA1537, TA1535 and WP2uvrA in the presence of the test solution compared with the mean of vehicle control value.The positive controls mean exhibited at least a 3-fold increase over the respective mean of the Salmonellatyphimurium and Escherichia coli tester strains used. The results are summarized in Appendix 2.

VII. Conclusion

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that under the conditions of this assay, the test article solution was considered to be Non-Mutagenic to Salmonella typhimurium tester strains TA98, TA100, TA1537, TA1535 and Escherichia coli WP2uvrA.The negative and positive controls performed as anticipated. The results of this study should be evaluated in conjunction with other required tests as listed in ISO 100993, Part 3: Tests for Genotoxicity, Carcinogenicity, and Reproductive Toxicology.




Page 4 of 8


Appendix 2:

Bacterial Mutation Assay Plate Incorporation Assay Results

Concentration µg per Plate

TA98

Revertants per plate (CFU) Mean

Test Solution w/ S9

5000 21 25 23

1500 21 32 27

500 45 48 47

150 35 34 35

50 48 37 43

15 39 55 47

5.0 42 44 43

1.5 32 31 32

Test Solution w/o S9

5000 15 25 20

1500 32 44 38

500 29 34 32

150 36 18 27

50 20 15 18

15 37 36 37

5.0 33 25 29

1.5 12 16 14

DI Water w/S9 44 36 40

DI Water w/o S9 35 47 41

2-aminoanthracen w/ S9 398 364 381

2-nitrofluorene w/o S9 145 127 136

Historical Count Positive w/S9 43-1893

Historical Count Positive w/o S9 39-1871

Historical Count Negative w/S9 4-69

Historical Count Negative w/o S9 3-59

*CFU = Colony Forming Units*Mean = Average of duplicate plates




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TA100 Revertants per plate

(CFU) Mean

Test Solution w/ S9

5000 28 35 32

1500 111 106 109

500 102 98 100

150 103 105 104

50 92 95 94

15 87 86 87

5.0 99 99 96

1.5 112 115 114


5000 26 35 31

1500 45 44 45

500 63 65 64

150 72 64 68

50 45 65 55

15 88 84 86

5.0 87 86 87

1.5 115 89 102

DI Water w/S9 102 117 110

DI Water w/o S9 95 111 103


Sodium azide w/o S9 645 676 661









Page 6 of 8




(CFU) Mean

Test Solution w/ S9

5000 4 5 5

1500 11 15 13

500 12 14 13

150 10 11 11

50 15 15 15

15 12 14 13

5.0 10 10 10

1.5 8 9 9


5000 2 8 5

1500 6 2 4

500 8 10 9

150 12 16 14

50 10 14 12

15 11 14 13

5.0 13 15 14

1.5 9 13 11




2-aminoacridine w/o S9 512 550 531









Page 7 of 8




(CFU) Mean

Test Solution w/ S9

5000 15 13 14

1500 12 13 13

500 15 14 15

150 12 16 14

50 8 19 14

15 11 10 11

5.0 12 21 17

1.5 15 13 14


5000 15 2 9

1500 12 16 14

500 11 17 14

150 18 16 17

50 10 15 13

15 17 19 18

5.0 13 14 14

1.5 11 16 14




Sodium azide w/o S9 577 583 580









Page 8 of 8



WP2uvrA Revertants per plate

(CFU) Mean

Test Solution w/ S9

5000 35 32 34

1500 42 25 34

500 27 28 28

150 32 34 33

50 44 16 30

15 28 31 30

5.0 38 45 42

1.5 35 37 36


5000 21 25 23

1500 26 29 28

500 44 47 46

150 25 31 28

50 35 23 29

15 26 31 29

5.0 34 33 34

1.5 31 36 34




Methylmethanesulfonate w/o S9 301 312 307








Phototoxicity Assay Analysis 107 Technology Drive Lincolnton, NC 28092

(704) 276-7100 Fax (704) 276-7101

Page 1 of 4


Code: M16001

CAS #: 68606-81-5


Lot #: 41416P


Test Performed: In Vitro EpiDerm™ Model (EPI-200-SIT) Phototoxicity

SUMMARY

In vitro phototoxicity irritation studies were conducted to evaluate whether PhytoCide Black Currant Powder would induce phototoxic irritation in the EpiDerm™ model assay.

The product was tested according to the manufacturer’s protocol. The test article solution was found to be a non-photoirritant at concentrations of 0.5%, 1.3%, and 4.5%. Reconstructed human epidermis was incubated in growth media for one hour to allow for tissue equilibration after shipping from MatTek Corporation, Ashland, MA. Test substance was applied to the tissue inserts in five varying concentrations and incubated overnight at 37°C,5% CO2, and 95% relative humidity (RH). The following day, the appropriate tissue inserts were irradiated (UVA) for 60 minutes with 1.7 mW/cm2 (=6 J/cm2). After substance incubation, irradiation, and washing was completed, the cell viability test was conducted. Cell viability was measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl)], present in the cell mitochondria, into blue formazan salt that was measured after extraction from the tissue. The photoirritation potential of the test chemical was dictated by the reduction in tissue viability of UVA exposed tissues compared to non-UVA exposed tissues.

Under the conditions of this assay, the test article was considered to be non-phototoxic at concentrations of 0.5%, 1.3%, and 4.5%. The negative and positive controls performed as anticipated.

I. Introduction

A. PurposeIn vitro dermal phototoxicity study was conducted to evaluate whether a test article would inducephotoirritation in the EpiDerm™ model assay. MatTek Corporation’s reconstructed human epidermal modelis becoming a standard in determining the phototoxicity potential of a test substance. This assay is able todiscriminate between photoirritants and non-photoirritants at varying concentrations.




(704) 276-7100 Fax (704) 276-7101

Page 2 of 4

II. Materials

A. Incubation Conditions: 37°C at 5% CO2 and 95% relative humidityB. Equipment: Forma humidified incubator, ESCO biosafety laminar flow hood, Synergy

HT Microplate reader; UVA-vis Irradiation Equipment; UVA meter; Pipettes

C. Media/Buffers: Dulbecco’s Modified Eagle Medium (DMEM) based medium; Dulbecco’s Phosphate-Buffered Saline (DPBS); sterile deionized H2O

D. Preparation: Pre-incubate (37°C) tissue inserts in assay medium; Place assay mediumand MTT diluent at 4°C, MTT concentrate at -20°C, and record lotnumbers of kit components

E. Tissue Culture Plates: Falcon flat bottom 96-well, 24-well, and 6-well tissue culture plates F. Reagents: MTT (3-4,5-dimethyl thiazole 2-yl) (1.0mg/mL); Extraction Solution

(Isopropanol); Chlorpromazine; Triton X-100 (1%) G. Other: Wash bottle; sterile disposable pipette tips; Parafilm; forceps

III. Test Assay

A. Test SystemThe reconstructed human epidermal model, EpiDerm™ consists of normal human-derived epidermalkeratinocytes which have been cultured to form a multilayer, highly differentiated model of the humanepidermis. This model consists of organized basal, spinous, and granular layers, and contains a multilayerstratum corneum containing intercellular lamellar lipid layers. The EpiDerm™ tissues are cultured onspecially prepared cell culture inserts.

B. Negative ControlSterile deionized water is used as the negative controls for the EpiDerm™ Phototoxicity assay.

C. Positive ControlConcentrations of chloropromazine, ranging from 0.001% to 0.1%, were used as positive controls for theEpiDerm™ Phototoxicity assay.

D. Data Interpretation ProcedureA photoirritant is predicted if the mean relative tissue viability of the 2 tissues exposed to the test substanceand 60 minutes of 6 J/cm2 is reduced by 20% compared to the non-irradiated control tissues.

IV. Method

A. Tissue ConditioningUpon MatTek kit arrival at Active Micro Technologies, LLC the tissue inserts are removed from theirshipping medium and transferred into fresh media and tissue culture plates and incubated at 37°C at 5%CO2 and 95% relative humidity for 60 minutes. After those 60 minutes the inserts are transferred into freshmedia and tissue culture plates and tissue insert dosing begins.

B. Test Substance Exposure50µL of the diluted test substance in their respective concentrations are applied to 2 tissue inserts andallowed to incubate for overnight in a humidified incubator (37°C, 5% CO2, 95% RH).




(704) 276-7100 Fax (704) 276-7101

Page 3 of 4

C. Tissue IrradiationTissue inserts in their 6-well plates are UVA-irradiated for 60 minutes with 6 J/cm2 at room temperature.The non-irradiated tissue inserts are incubated at room temperature in the dark.

D. Tissue Washing and Post IncubationAfter UVA-irradiation and dark incubation is complete the tissue inserts are washed using sterile DPBS andtransferred to fresh 6-well plates and media for overnight incubation at 37°C, 5% CO2, 95% RH.

E. MTT AssayTissue inserts are transferred into 300µL MTT media in pre-filled plates and incubated for 3 hours at 37°C,5% CO2, and 95% RH. Inserts are then removed from the MTT medium and placed in 2mL of theextraction solution. The plate is sealed and incubated at room temperature in the dark for 24 hours. Afterextraction is complete the tissue inserts are pierced with forceps and 2 x 200µL aliquots of the blueformazan solution is transferred into a 96 well plate for Optical Density reading. The spectrophotometerreads the 96-well plate using a wavelength of 570 nm.

V. Acceptance Criterion

A. Negative ControlThe results of this assay are acceptable if the mean negative control Optical Density (OD570) is ≥ 0.8.

B. Positive ControlThe assay meets the acceptance criterion if a dose dependent reduction in cell viability in the UVA-irradiated tissues is between 0.00316% and 0.0316%.

C. Standard DeviationSince the phototoxicity potential is predicted from the mean viability of 2 tissues for the EpiDerm™Phototoxicity Protocol, the variability of the replicates should not exceed 30%.

VI. Results

A. Tissue CharacteristicsThe tissue inserts included in the MatTek EpiDerm™ assay kit were in good condition, intact, and viable.

B. Tissue Viability AssayThe results are summarized in Figure 1. Cell viability is calculated for each tissue as a percentage of thecorresponding vehicle control either irradiated or non-irradiated. Tissue viability was not reduced by 20% inthe presence of the test substance and UVA-irradiation at concentrations of 0.5%, 1.3%, and 4.5%. Thenegative control mean exhibited acceptable relative tissue viability while the positive control exhibited dosedependent loss of tissue viability and cell death.

C. Test ValidityThe data obtained from this study met criteria for a valid assay. The negative and positive controls performedas anticipated.




(704) 276-7100 Fax (704) 276-7101

Page 4 of 4

VII. ConclusionPhototoxicity (photoirritation) is defined as an acute toxic response that is elicited after exposure of the skin tocertain chemicals and subsequent exposure to light. Under the conditions of this assay, the test articlesubstance was considered to be non-phototoxic at concentrations of 0.5%, 1.3%, and 4.5%. There is adecrease in viability at the 12% test concentration with and without irradiation. Using any test substance at thishigh of a concentration will have noticeable effects on cellular viability due to the fact that that substance isreplacing the cell’s nutrients. We can safely say that PhytoCide Black Currant Powder is not a photoirritantwhen used at the suggested use levels of 1 – 3%.

Figure 1: EpiDerm Phototoxicity Graph

0.5% 1.3% 4.5% 12%Control PhytoCide Black Currant Powder

Without Irradiation 100 97.1 96.5 100.9 75.7With Irradiation 100 102 98.5 94.1 50.2

0

20

40

60

80

100

120

Tiss

ue V

iabi

lity

(% U

ntre

ated

Con

trol

)

EpiDerm Phototoxicity Assay M16001 PhytoCide Black Currant Powder Without Irradiation

With Irradiation



OECD 202 Acute Daphnia Assay


Page 1 of 7


Code: M16001

CAS #: 68606-81-5


Lot #: 39836P


Test Performed: OECD 202 Daphnia spp. Acute Immobilization Test

Introduction

The purpose of the present study is to determine the toxicity of PhytoCide Black Currant Powder by exposing Daphnia spp. to the test substance for 48 hours and measuring the immobilization rate against the control. The present study defines an organism as being immobilized when it does not move for 15 seconds after the test vessel is gently shaken.

OECD Guideline 202 on “Daphnia spp., Acute Immobilization Test and Reproduction Test”, adopted in 1984, included two parts: Part I – the 24 hour EC50 acute immobilization test and Part II – the reproduction test (at least 14 days). Revision of the reproduction test resulted in the adoption and publication of Test Guideline 211 on “Daphnia magna Reproduction Test” in September 1998. Consequently, the new version of Guideline 202 is restricted to the acute immobilization test.

Assay Principle

Young daphnids, aged less than 24 hours at the start of the test, are exposed to the test substance at a range of concentrations for a period of 48 hours. Immobilization is recorded at 24 hours and 48 hours and compared with control values. The results are analyzed in order to calculate the EC50 at 48 hours. EC50 is the concentration estimated to immobilize 50% of the daphnids within a stated exposure period. Immobilization refers to those animals that are not able to swim within 15 seconds after gentle agitation of the test vessel, even if they can still move their antennae.

The water solubility and vapor pressure of the test substance should be known. A reliable analytical method for the quantification of the substance in the test solutions with reported recovery efficiency and limit of determination should also be available.





Page 2 of 7

A reference substance may be tested for EC50 as a means of assuring that the test conditions are reliable.

For this assay to be valid, the following performance criteria apply: • In the control, not more than 10% of the daphnids should have been immobilized.• The dissolved oxygen concentration at the end of the test should be at least 3 mg/L in control and test

vessels.

Materials

• Glass Test Tubes and/or Beakers• Dissolved Oxygen Meter• pH Meter• Temperature Control Apparatus• Total Organic Carbon (TOC) Analyzer• Chemical Oxygen Demand (COD) Analyzer• Daphnia magna Straus

o Use organisms less than 24 hours old. Do not use first offspring of parents.• Water

o Use water suitable for culturing and testing Daphnia spp. It can be natural water (surfacewater or groundwater), dechlorinated tap water, or artificially prepared water (Table 1), but mustsatisfy the conditions listed in Table 2. Do not use Elendt M4 or M7 media or water containingchelating agents for testing metal-containing substances. The water hardness should be 250 mg/Lor smaller in terms of calcium carbonate concentration, and the pH should be 6-9. Aerate thematerial water before using it for the test.

Substance Concentration Particulate Matter <20 mg/L Total Organic Carbon <2 mg/L Unionized Ammonia <1 ug/L Residual Chlorine <10 ug/L Total Organophosphorus Pesticides <50 ng/L Total Organochlorine Pesticides plus Polychlorinated Biphenyls

<50 ng/L

Total Organic Chlorine <25 ng/L Table 1: Chemical Characteristics of Suitable Water

Table 2: Examples of Suitable Reconstituted Test Water

Substance Amount Added to 1 Liter Water

To prepare the reconstituted water, add the following volumes of stock solutions to 1 liter

water Calcium Chloride 11.76 grams 25 mL Magnesium Sulfate 4.93 grams 25 mL Sodium Bicarbonate 2.59 grams 25 mL Potassium Chloride 0.23 grams 25 mL





Page 3 of 7

Methods

Test Conditions • Test Method

o Test is performed under a static, semi-static, or flow-through condition. If test substance isunstable, a semi-static or flow-through test is recommended.

• Exposure Periodo 48 hours

• Test Volumeo At least 2 milliliters

• Number of Test Organismso At least 20 organisms for each test concentration and the control.

• Test Concentrationo Adopt a concentration range of at least 5 concentrations, with the highest concentration inducing

100% immobilization and no effect at the lowest concentration.• Culture Method

o Illumination: The photoperiod is set to 16 hours light and 8 hours darko Temperature: The temperature is between 18°C to 22°Co Dissolved Oxygen Concentration: Must be kept at 3mg/L or highero Feeding: Do not feed test organisms

Observation • Observe mobility of the organisms at least twice (i.e., at 24 and 48 hours after exposure).• The organisms are considered immobilized when they do not move for 15 seconds after test vessel is

gently shaken.

Measurement of Test Substance Concentrations • At the beginning and end of exposure, measure test substance concentrations at the lowest and highest

test concentration groups.o For volatile or adsorptive substances, additional measurements are recommended at 24 hours

intervals during exposure period.

Test Condition Measurements • Measure dissolved oxygen in the control and at the highest test concentration at the beginning and end

of the exposure period.• Measure pH in the control and at the highest test concentration at the beginning and end of the exposure

period.• Water temperature should be measured at the beginning and end of the exposure period.





Page 4 of 7

Data and Reporting

I. Dataa. Data should be summarized in tabular form, showing for each treatment group and control, the number of

daphnids used, and immobilization at each observation. The percentages immobilized at 24 and 48 hoursare plotted against test concentrations. Data are analyzed by appropriate statistical methods (e.g. probitanalysis, etc.) to calculate the slopes of the curves and the EC50 with 95% confidence limits (p = 0.95).

b. Where the standard methods of calculating the EC50 are not applicable to the data obtained, the highestconcentration causing no immobility and the lowest concentration producing 100% immobility should beused as an approximation for the EC50 (this being considered the geometric mean of these twoconcentrations).

II. Test Reporta. The test report must include the following:

i. Test substance:1. Physical nature and relevant physical-chemical properties2. Chemical identification data, including purity

ii. Test species:1. Source and species of Daphnia, supplier of source (if known), and the culture conditions

(including source, kind and amount of food, feeding frequency)iii. Test conditions:

1. Description of test vessels: type and volume of vessels, volume of solution, number ofdaphnids per test vessel, number of test vessels (replicates) per concentration

2. Methods of preparation of stock and test solutions including the use of any solvent ordispersants, concentrations used

3. Details of dilution water: source and water quality characteristics (pH, hardness, Ca/Mgratio, Na/K ratio, alkalinity, conductivity, etc); composition of reconstituted water if used

4. Incubation conditions: temperature, light intensity and periodicity, dissolved oxygen, pH,etc.

iv. Results:1. The nominal test concentrations and the result of all analyses to determine the

concentration of the test substance in the test vessels; the recovery efficiency of themethod and the limit of determination should also be reported

2. All physical-chemical measurements of temperature, pH and dissolved oxygen madeduring the test

3. The EC50 at 48 hours for immobilization with confidence intervals and graphs of the fittedmodel used for calculation, the slopes of the dose-response curves and their standarderror; statistical procedures used for determination of EC50





Page 5 of 7

Results

General Information:

Name of new chemical substance PhytoCide Black Currant Powder

INCI Nomenclature Ribes nigrum (Black Currant) Fruit Extract

CAS number 68606-81-5 Structural or rational formula (if neither is

available, summarize its formulation method)

Botanical: Ribes nigrum fruit

Molecular weight 242.5 Daltons Purity of the new chemical substance

used for the test (%) 100%

Lot number of the new chemical substance used for the test 39836P

Names and contents of impurities n/a Solubility in water Soluble up to 3% in solution

Melting point n/a Boiling point n/a

Properties at room temperature Free flowing light pink powder Stability Stable under ordinary conditions

Solubility in solvents, etc. Solvent Solubility Stability in solvent

n/a n/a n/a





Page 6 of 7

Test Materials and Methods: Items Contents

Test Organisms

Species Daphnia magna

Source Carolina Biological Supply Company

Susceptibility to reference substance (EC50)

Potassium dichromate (0.94 mg/L)

Culture Kind of Medium Elendt Medium M4

Conditions (Temperature/Photoperiod) 20°C/16 Hour Light-8 Hour Dark

Test Conditions

Test Vessel Glass

Material Water Kind Elendt Medium M4

Hardness 250 mg/L pH 7.4

Date of Exposure 1/20/2015 Test Concentrations 200, 90.9, 41.3, 18.8, 8.5 mg/L

Number of organisms 120 Number of Replicates

Exposure Group 4 Control Group 4

Test Solution Volume 2 mL

Vehicle

Use or Not N/A Kind N/A

Concentration N/A Number of Replicates N/A

Culture Method (Static, Semi-Static, Flow-Through) Static

Water Temperature 20°C ± 2°CDissolved Oxygen Concentration (DO) 3 mg/L

Photoperiod 16 Hour Light-8 Hour Dark Calculation of

Results Statistical Method Probit Analysis





Page 7 of 7

Test Results: Items Contents

Toxicity Value 48hr EC50 130.7 mg/L Exposure Concentrations

Used for Calculation Nominal Values 200, 90.9, 41.3, 18.8, 8.5 mg/L

Remarks Not harmful to aquatic organisms

Discussion

After 48 hours, the EC50 value for PhytoCide Black Currant Powder was determined to be 130.7 mg/L. The conditions of OECD guideline 202 for the validity of the test were adhered to: The immobility of controls in purified drinking water (dilution water) did not exceed 10%. According to the EU Directive 93/67/EEC, this product is not classified and therefore not harmful to aquatic organisms.



OECD 301B Ready Biodegradability Assay


Page 1 of 6


Code: M16001

CAS #: 68606-81-5


Lot #: 39441P

Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092

Study Director: Erica Segura

Principle Investigator: Meghan Darley

Test Performed: OECD 301 B Ready Biodegradability: CO2 Evolution (Modified Sturm Test)

Introduction

A study was conducted to assess the ready biodegradability of PhytoCide Black Currant Powder in an aerobic aqueous medium. In the OECD guideline 301 for ready biodegradability, six methods are provided as options. This report uses method B, CO2 Evolution, also known as a Modified Sturm Test. This method was chosen based on the solubility, volatility, and adsorbing capabilities of the test sample.

Assay Principle

A solution or suspension of the test substance in a mineral medium is inoculated and incubated under aerobic conditions in the dark or in diffuse light. The amount of DOC (Dissolved Organic Carbon) in the test solution due to the inoculum should be kept as low as possible compared to the amount of organic carbon due to the test substance. Allowance is made for the endogenous activity of the inoculum by running parallel blanks with inoculum but without test substance. A reference compound is run in parallel to check the procedures’ operation.

In general, degradation is followed by the determination of parameters such as DOC, carbon dioxide production, and oxygen uptake. Measurements are taken at sufficiently frequent intervals to allow the identification of the beginning and end of biodegradation.

Normally this test lasts for 28 days, but it may be ended before that time if the biodegradation curve reaches a plateau for at least three determinations. Tests may also be prolonged beyond 28 days when the curve shows that biodegradation has started but the plateau has not yet been reached. In such cases the test substance would not be classified as readily biodegradable.





Page 2 of 6

The pass levels for ready biodegradability are 70% removal of DOC and 60% of ThOD (Theoretical Oxygen Demand) or ThCO2 (Theoretical Carbon Dioxide) production for respirometric methods. They are lower in the respirometric methods since, as some of the carbon from the test chemical is incorporated into new cells, the percentage of CO2 produced is lower than the percentage of carbon being used. These pass values have to be reached in a 10-day window within the 28-day period of the test. The 10-day window begins when the degree of biodegradation has reached 10% DOC, ThOD, or ThCO2 and must end before day 28 of the test. Test substances which reach the pass levels after the 28-day period are not deemed to be readily biodegradable.

In order to check the procedure, reference compounds which meet the criteria for ready biodegradability are tested by setting up an appropriate vessel in parallel as part of normal test runs. Suitable compounds are freshly distilled aniline, sodium acetate, and sodium benzoate. These compounds all degrade in this method even when no inoculum is deliberately added.

Because of the nature of biodegradation and of the mixed bacterial populations used as inocula, determinations should be carried out at least in duplicate. It is usually found that the larger the concentration of microorganisms initially added to the test medium, the smaller the variation between replicates.

Materials Water

o Deionized or distilled, free from inhibitory concentrations of toxic substanceso Must contain no more than 10% of the organic carbon content introduced by the test materialo Use only one batch of water for each series of tests

Mineral mediao To prepare the mineral medium, mix 10 mL of solution A with 800 mL water. Then add 1 mL each of

solutions B, C, and D and make up to 1 liter with water.o Solution A (Dissolve in water and make up to 1 liter; pH 7.4)

Potassium dihydrogen orthophosphate, KH2PO………………..……………………………….....8.5g Dipotassium hydrogen orthophosphate, K2HPO4………………………………………….21.8g Disodium hydrogen orthophosphate dehydrate, Na2HPO4.2H2O....................................33.4g Ammonium chloride, NH4Cl…………………………………………………………………....0.5g

o Solution B (Dissolve in water and make up to 1 liter) Calcium chloride, anhydrous, CaCl2………………………………………………….……27.50g

Or Calcium chloride dehydrate, CaCl2.2H2O…………………………………………………36.40g

o Solution C (Dissolve in water and make up to 1 liter) Magnesium sulphate heptahydrate, MgSO4.7H2O……………………………………… 22.50g

o Solution D (Dissolve in water and make up to 1 liter.) Iron (III) chloride hexahydrate, FeCl3.6H2O………………………………………………...0.25g

o Flasks, 2-5 liters each, fitted with aeration tubes reaching nearly to the bottoms of the vessels and anoutlet

o Magnetic stirrerso Gas absorption bottleso Device for controlling and measuring air flowo Apparatus for carbon dioxide scrubbing, for preparation of air which is free from carbon dioxide;

alternatively, a mixture of CO2-free oxygen and CO2-free nitrogen from gas cylinders in the correctproportions (20% O2 : 80% N2)

o Device for determination of carbon dioxide, either titrimetrically or by some form of inorganic carbonanalyzer





Page 3 of 6

o Stock solutions of test substances When solubility of the substance exceeds 1 g/L, dissolve 1-10 g, as appropriate, of test or

reference substance in water and make up to 1 liter. Otherwise, prepare stock solutions inmineral medium or add the chemical directly to the mineral medium, making sure it

o Inoculum The inoculum may be derived from the following sources

Activated sludge

Sewage effluents

Surface waters

Soils

Or from a mixture of these.

Inoculum may be pre-conditioned to the experimental conditions, but not pre-adapted to thetest substance. Pre-conditioning consists of aerating activated sludge in mineral medium orsecondary effluent for 5-7 days at the test temperature. Pre-conditioning sometimes improvesthe precision of the test method by reducing blank values.

Methods

I. Preparation of flasks: As an example, the following volumes and weights indicate the values for 5-liter flaskscontaining 3 liters of suspension. If smaller volumes are used, modify the values accordingly.

a. To each 5-liter flask, add 2,400 mL mineral medium.b. Add an appropriate volume of the prepared activated sludge to give a concentration of suspended

solids of not more than 30 mg/L in the final 3 liters of inoculated mixture. Alternatively, first dilute theprepared sludge to give a suspension of 500-1000 mg/L in the mineral medium before adding analiquot to the contents of the 5-liter flask to attain a concentration of 30 mg/L.

c. Aerate these inoculated mixtures with CO2-free air overnight to purge the system of carbon dioxide.d. Add the test material and reference compound, separately, as known volumes of stock solutions, to

replicate flasks to yield concentrations, contributed by the added chemicals, of 10 – 20 mg DOC orTOC per liter. Leave some flasks without addition of chemicals as inoculum controls. Add poorlysoluble test substances directly to the flasks on a weight or volume basis. Make up the volumes ofsuspensions in all flasks to 3 liters by the addition of mineral medium previously aerated with CO2-free air.

e. If required, use one flask to check the possible inhibitory effect of the test substance by adding boththe test and reference substances at the same concentrations as present in the other flasks.

f. If required, check whether the test substance is degraded abiotically by using a sterilizeduninoculated solution of the chemical. Sterilize by the addition of a toxic substance at an appropriateconcentration.

g. If barium hydroxide is used, connect three absorption bottles, each containing 100 mL of 0.0125Mbarium hydroxide solution, in series to each 5-liter flask. The solution must be free of precipitatedsulfate and carbonate and its strength must be determined immediately before use.

h. If sodium hydroxide is used, connect two traps, the second acting as a control to demonstrate that allthe carbon dioxide was absorbed in the first. Absorption bottles fitted with serum bottle closures aresuitable. Add 200 mL 0.05M sodium hydroxide to each bottle. This is sufficient to absorb the totalquantity of carbon dioxide evolved when the test substance is completely degraded.

i. In a typical run, the following flasks are used:i. Flasks 1 & 2: containing test substance and inoculum (test suspension)ii. Flasks 3 & 4: containing only inoculum (inoculum blank)iii. Flask 5: containing reference compound and inoculum (procedure control)iv. Flask 6: containing test substance and sterilizing agent (abiotic sterile control)v. Flask 7: containing test substance, reference compound and inoculum (toxicity control)





Page 4 of 6

II. Start the test by bubbling CO2-free air through the suspensions at a rate of 30-100 mL/minute.

III. CO2 Determinationa. It is mandatory to follow the CO2 evolution from the test suspensions and inoculum blanks in parallel

and it is advisable to do the same for the other test vessels.b. During the first ten days it is recommended that analyses of CO2 should be made every second or

third day and then at least every fifth day until the 28th day so that the 10-day window period can be

identified. On the days of CO2 measurement, disconnect the barium hydroxide absorber closest to thetest vessel and titrate the hydroxide solution with 0.05M HCl using phenolphthalein as the indicator.Move the remaining absorbers one place closer to the test vessel and place a new absorbercontaining 100 mL fresh 0.0125M barium hydroxide at the far end of the series. Make titrations areneeded (for example, when substantial precipitation is seen in the first trap and before any is evidentin the second, or at least weekly). Alternatively, with NaOH as absorbent, withdraw a sample of thesodium hydroxide solution from the absorber nearest to the test vessel using a syringe. The samplevolume needed will depend on the carbon analyzer used, but sampling should not significantlychange the absorbent volume over the test period. Inject the sample into the IC part of the carbonanalyzer for analysis of evolved carbon dioxide directly. Analyze the contents of the second trap onlyat the end of the test in order to correct for any carry-over of carbon dioxide.

c. On the 28th day withdraw samples, optionally, for DOC and/or specific chemical analysis. Add 1 mL of

concentrated hydrochloric acid to each test vessel and aerate them overnight to drive off the carbondioxide present in the test suspensions. On day 29 make the last analysis of evolved carbon dioxide.

Data and Reporting

I. Treatment of Resultsa. Data from the test should be entered onto the attached data sheet.b. The amount of CO2 produced is calculated from the amount of base remaining in the absorption bottle.

When 0.0125M Ba(OH)2 is used as the absorbent, the amount remaining is assessed by titrating with0.05M HCl.

c. Since 1 mmol of CO2 is produced for every mol of Ba(OH)2 reacted to BaCl2 and 2 mmol of HCl areneeded for the titration of the remaining Ba(OH)2 and given that the molecular weight of CO2 is 44 g,the weight of CO2 produced (in mg) is calculated by:

0.05 × (50 − 𝑚𝐿 𝐻𝐶𝑙 𝑇𝑖𝑡𝑟𝑎𝑡𝑒𝑑) × 44

2 = 1.1 × (50 − 𝑚𝐿 𝐻𝐶𝑙 𝑇𝑖𝑡𝑟𝑎𝑡𝑒𝑑)

Therefore, the factor to convert volume of HCl titrated to mg CO2 produced is 1.1 in this case. Calculate the weights of CO2 produced from the inoculum alone and from the inoculum plus test substance using the respective titration values. The difference is the weight of CO2 produced from the test substance alone.





Page 5 of 6

d. The percentage biodegradation is calculated from:

% 𝐷𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = 𝑚𝑔 𝐶𝑂2 𝑃𝑟𝑜𝑑𝑢𝑐𝑒𝑑

𝑇ℎ𝐶𝑂2 × 𝑚𝑔 𝑇𝑒𝑠𝑡 𝑆𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝐴𝑑𝑑𝑒𝑑 × 100

Or

% 𝐷𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = 𝑚𝑔 𝐶𝑂2 𝑃𝑟𝑜𝑑𝑢𝑐𝑒𝑑

𝑚𝑔 𝑇𝑂𝐶 𝐴𝑑𝑑𝑒𝑑 𝑖𝑛 𝑇𝑒𝑠𝑡 × 3.67 × 100

𝑊ℎ𝑒𝑟𝑒 3.67 𝑖𝑠 𝑡ℎ𝑒 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 (44

12) 𝑓𝑜𝑟 𝑐𝑎𝑟𝑏𝑜𝑛 𝑡𝑜 𝑐𝑎𝑟𝑏𝑜𝑛 𝑑𝑖𝑜𝑥𝑖𝑑𝑒

e. When NaOH is used as the absorbent, calculate the amount of CO2 produced after any time intervalfrom the concentration of inorganic carbon and the volume of absorbent used. Calculate thepercentage degradation from:

% 𝑇ℎ𝐶𝑂2 = 𝑚𝑔 𝐼𝐶 𝑓𝑟𝑜𝑚 𝑇𝑒𝑠𝑡 𝐹𝑙𝑎𝑠𝑘 − 𝑚𝑔 𝐼𝐶 𝑓𝑟𝑜𝑚 𝐵𝑙𝑎𝑛𝑘

𝑚𝑔 𝑇𝑂𝐶 𝐴𝑑𝑑𝑒𝑑 𝑎𝑠 𝑇𝑒𝑠𝑡 𝑆𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒𝑠 × 100

f. Display the course of degradation graphically and indicate the 10-day window. Calculate and report thepercentage removal achieved at the plateau, at the end of the test, and/or at the end of the 10-daywindow, whichever is appropriate.

g. When appropriate, calculate DOC removals using the equation given in 301 A paragraph 27.h. When an abiotic control is used, calculate the percentage abiotic degradation by:

% 𝐴𝑏𝑖𝑜𝑡𝑖𝑐 𝐷𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = 𝐶𝑂2 𝑃𝑟𝑜𝑑𝑢𝑐𝑒𝑑 𝑏𝑦 𝑆𝑡𝑒𝑟𝑖𝑙𝑒 𝐹𝑙𝑎𝑠𝑘 𝐴𝑓𝑡𝑒𝑟 28 𝐷𝑎𝑦𝑠 (𝑚𝑔)

𝑇ℎ𝐶𝑂2 (𝑚𝑔) × 100

Validity of Tests

I. The IC content of the test substance suspension in the mineral medium at the beginning of the test must beless than 5% of the TC, and the total CO2 evolution in the inoculum blank at the end of the test should notnormally exceed 40 mg/L medium. If values greater than 70 mg CO2/L are obtained, the data andexperimental technique should be examined critically.





Page 6 of 6

Data Sheet

Laboratory Active Concepts Tissue Culture Laboratory

Test Start Date 12/29/2014

Test Substance

Name PhytoCide Black Currant

Powder

Stock Solution Concentration 2 g/L

Initial Concentration in Medium 20 mg/L

Inoculum

Source Activated Sludge

Treatment Given Centrifugation

Pre-conditioning N/A

Suspended Solids Concentration in Reaction Mixture

4 mg/L

Reference Material Sodium Benzoate Concentration 20 mg/L

CO2 Production and Degradability

Method

Ba(OH)2 0.0125M

NaOH N/A

Other N/A

Total Contact Time 28 Days

Total CO2 Evolved Measurements

Days 2, 4, 11, 17, 23, 28

Degradation Over Time 92.5%

Remarks Test material was readily biodegradable

Conclusion This test met the criteria for a valid assay

Discussion

Based on the testing conducted in accordance with the specified method, test PhytoCide Black Currant Powder achieved 92.5% biodegradation after 28 days of testing. The product met method requirements for Readily Biodegradability classification.


Version#1/04-06-15


Date Issued: April 6, 2015

ALLERGEN DECLARATION

RE: PhytoCide Black Currant Powder (M16001)

Please be advised that this form is to certify that the above referenced product, manufactured at Active Micro Technologies, LLC, does not contain any of the allergens listed below:

Eggs – or egg products

Milk – or milk products (includes whey, lactose, casein, milk, cream)

Peanuts – or peanut products

Fish – (includes fish: surimi, cod, pollack, whitefish)

Shellfish – (shrimp, lobster, crab, clams, etc.)

Soybeans – or soybean products (includes soya powder, protein, oil, lecithin, tofu)

Wheat – or wheat products (includes Gluten)

Tree nuts – (almond, brazil nut, cashew, chestnut, hazelnut, filbert, pine nuts (pinyon, pinon), pistachio, pecan, macadamia, walnut).

Palm Oil – or palm kernel oil

If you have any further questions or concerns, please contact us at: 1-704-276-7100



Certificate of Origin 107 Technology Drive Lincolnton, NC 28092

(704) 276-7100 Fax (704) 276-7101


Active Micro Technologies, LLC certifies that all raw material(s) used to manufacture the above listed ingredient originate in the United States of America.

Active Micro Technologies, LLC certifies that the above listed ingredient is plant derived from non-GMO Ribes nigrum and therefore is BSE-Free.

Active Micro Technologies, LLC certifies that the above listed ingredient can be classified as Vegan Compliant.

Active Micro Technologies, LLC certifies that the above listed ingredient has never been tested on animals.




Date: 03 / 18 / 2015 Version: 4

Page: 1/9

Cancels and replaces version: 3

Safety Data Sheet 107 Technology Drive Lincolnton, NC 28092

(704) 276-7100 Fax (704) 276-7101

SECTION 1. IDENTIFICATION

PhytoCide Black Currant Powder M16001

Topical Cosmetic Use; Antimicrobial

Product Name/Identifier Product Code

Recommended Use Restrictions on Use None

Supplier/Manufacturing Site Active Micro Technologies, LLC Address 107 Technology Drive

Lincolnton, NC 28092, USA

Telephone No. (24hrs) 1-704-276-7100Fax No. 1-704-276-7101

Emergency Telephone # # 1-704-276-7100 (Mon-Fri: 8:00AM – 5:00PM EST)

SECTION 2. HAZARD(S) IDENTIFICATION

Classification:

Based on present data no classification and labeling is required according to GHS, taking into account the national implementation (United Nations version 2011)

This material is non-hazardous as defined by the American OSHA Hazard Communication Standard (29 CFR 1910.1200).

-According to present data no classification and labeling is requiredaccording to Directives 67/548/EEC or 1999/45/EC.-This product is not classified as hazardous to health or environmentaccording to the CLP regulation.

GHS/ CLP Basis for Classification:

USA OSHA Regulatory Status:

Europe Basis for Classification:

Labeling Elements: Pictograph: No hazard symbol expected

Hazard statements/Signal Word:d: Not applicable

Precautionaryystatements: P233: Keep container tightly closed P281: Use personal protective equipment as required P402: Store in a dry place P404: Store in a closed container P410: Protect from sunlight P411: Store at temperatures not exceeding 25°C




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(704) 276-7100 Fax (704) 276-7101

No particular hazards.

Other hazards which do not result in classification:

No particular fire or explosion hazard. By mechanical effect: By hydroscopic effect: No particular hazards.

US NFPA 704((National Fire Protection Association) Hazard Rating System:

Health hazard: Rating 0; Normal Material Flammability: Rating 0, Will Not Burn Reactivity: Rating 0, Stable Other Hazard Information: None

Results of PBT anddvPvB assessment:

-PBT: Not applicable-vPvB: Not applicable

SECTION 3. COMPOSITION / INFORMATION ON INGREDIENTS

Ribes Nigrum (Black Currant) Fruit Extract

Plant Extract

Common Chemical Name:

Generic name:

Chemical Familyy:

Description: Substance

Substance CAS Numbers EC Numbers Percentage Ribes Nigrum (Black Currant) Fruit Extract 68606-81-5 271-749-0 100.00%

Formula: Not applicable

SECTION 4. FIRST-AID MEASURES

In all cases of doubt, or when symptoms persist, seek medical attention. General:

Inhalation: Move to fresh air from exposure area. Get medical attention for any breathing difficulty.

Rinse with soap and water. Get medical advice if irritation develops. Skin contact:

Eye contact: Immediately rinse with water for at least 15 minutes, while keeping the eyes wide open. Consult with a physician.

Consult with a physician. Ingestion:

Protection of first-aiders: No special protection required.




Date: 03 / 18 / 2015 Version: 4

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(704) 276-7100 Fax (704) 276-7101

SECTION 5. FIRE-FIGHTING MEASURES

Not considered to be a fire and explosion hazard

Water, dry chemicals, foam & carbon dioxide.

None known

Move container from fire area if it can be done without risk. Avoid inhalation of material or combustion by-products. Stay upwind and keep out of low area

Boots, gloves, goggles.

SECTION 6. ACCIDENTAL RELEASE MEASURES

Avoid contact with eyes.

Personal Protective Equipment: -Protective goggles

Prevent entry into sewers and waterways. Do not allow material to contaminate ground water system

Pick up free liquid for recycling or disposal. Residual liquid can be absorbed on an inert material.

Wash non-recoverable remainder with water.

For disposal of residues refer to sections 8 & 13.

SECTION 7. HANDLING AND STORAGE

Labeling: Keep out of the reach of children. For industrial use, only as directed. Wash hands after use. Avoid storage near feed or food stuff.

Handling

Technical measures: Measures: Safe handling advice:

Storage Technical measures: Keep container closed. Recommended Storage Conditions: Store in a cool, dry place. This product should be stored at room temperature

(23 - 25°C). It should not be exposed to excessive heat or cold. Do not freeze.

Fire and explosion hazards::

Extinguishing media:

Suitable:

Not suitable:

Fire fighting:

Protection for fire-fighters:

Personal precautions:

Environmental precautions::

Methods for cleaning up:

Recovery:

Cleaning/Decontamination:

Disposal:




Date: 03 / 18 / 2015 Version: 4

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(704) 276-7100 Fax (704) 276-7101

Incompatible products: Avoid contact with strong oxidizers. Refer to the detailed list of incompatible materials (Section 10 Stability/Reactivity)

Packaging: Product may be packaged in normal commercial packaging. Packaging materials: Recommended - Polypropylene & High Density Polyethylene

SECTION 8. EXPOSURE CONTROLS / PERSONAL PROTECTION

Ensure adequate ventilation

Not Determined Not DeterminedNot Determined Not Determined

Not Determined

Precautionary statements:

Control parameters

Occupational exposure Limits:

France: ACGIH: Korea: UK:

Surveillance procedures: Engineering measures: Not Determined

Local exhaust Protective gloves made of rubber or neoprene. Safety glasses. Eye fountain. Suitable protective clothing

Personal Protective Equipment:

Respiratory protection: Hand protection: Eye protection: Collective emergency equipment: Skin and Body Protection:

Hygiene measures: Handle in accordance with good industrial hygiene and safety practice.

Measures related to the Environment: No particular measures.

SECTION 9. PHYSICAL AND CHEMICAL PROPERTIES

Free flowing powder Light pink with darker specks

Characteristic

3.0 – 6.0

8.0% Maximum

Partially water soluble

Appearance: Color:

Odor:

pH (1% Solution in Water):

Loss on Drying (1g-1hr-105°C):

Solubility (in Water):




Date: 03 / 18 / 2015 Version: 4

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(704) 276-7100 Fax (704) 276-7101

Citric Acid: 1.5 – 4.0%

Heavy Metals (Total): < 20 ppm Lead: < 10 ppm Arsenic: < 3 ppm Mercury: < 1 ppm

Specific Gravity: Not determined

Vapor density: Not applicable Boiling Point: Not applicable Freezing Point: Not applicable Melting point: Not determined

Flash point: Not applicable Oxidizing properties: Non oxidizing material according to EC criteria.

Solubility: In water: Partially soluble In organic solvents: Not determined Log P: Not determined

SECTION 10. STABILITY AND REACTIVITY

Stability: Stable under ordinary conditions of use and storage up to one year then re-test to full product specifications to extend shelf life

Hazardous reactions: None known

Conditions to avoid: No dangerous reactions known under use of normal conditions. Avoid extreme heat.

Materials to avoid: No dangerous reaction known with common products.

Hazardous decomposition products: None known

SECTION 11. TOXICOLOGICAL INFORMATION

Ingestion: Not Determined Dermal: Non-Irritant (Dermal Irritection Model) Ocular: Non-Irritant (Ocular Irritection Model) Inhalation: Not Determined

Acute toxicity data: EC50 (Acute Daphnia): 130.7 mg/L - Not harmful to aquatic organisms

Sensitization: Non-Primary Sensitizer




Date: 03 / 18 / 2015 Version: 4

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(704) 276-7100 Fax (704) 276-7101

No known effects Not Determined

Repeated dose toxicityy: Subacute to chronic toxicity: Mutagenicity/genotoxicityy: Non-mutagenic

Additional Toxicological Information: This product is not subject to classification according to the calculation method of the General EU Classification Guidelines for Preparations as issued in the latest version.

No known effects No known effects No known effects No known effects

Specific effects:

Carcinogenicity: Mutagenicity: Reproductive toxicity: Neuro-toxicity:

For more information: Does not present any particular risk on handling under normal conditions of good occupational hygiene practice.

This product has not been tested for the following: -Primary cutaneous and corrosive irritation-Acute oral toxicity

SECTION 12. ECOLOGICAL INFORMATION

Ecotoxicity Effects on the aquatic environment: Not Determined

Not Determined Biodegradability: Persistence:

Bioaccumulation: Octanol / water partition coefficient: Not Determined

Ultimate destination of the product: Soil & sediment.

Mobility:y: Precipitation: Expected behavior of the product:

Other Adverse Effects: None known

SECTION 13. DISPOSAL CONSIDERATIONS

Residues from product

Prohibition: Do not allow the product to be released into the Environment. Destruction/Disposal: Dispose of in accordance with relevant local regulations




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(704) 276-7100 Fax (704) 276-7101

Cleaning is not required prior to disposal.

Contaminate packaging

Decontamination/cleaning: Destruction/Disposal:

Note: Take all necessary precautions when disposing of this product according to local regulations.

SECTION 14. TRANSPORT INFORMATION

None None

Not classified as dangerous for transport

Material is not restrictive for land transport and is not regulated by ADR/RID Material is not restrictive for sea transport and is not regulated by IMO/IMDG Material is not restrictive for land transport and is not regulated by ICA/IATA

UN Number: UN Shipping Name:

Transport Hazard Class:

Land (rail/road): Sea: Air:

Marine Pollutant: No

Transport/Additional IInformation: Not regulated for US DOT Transport in non-bulk containers This material is not dangerous or hazardous

Special Precautions for User: None known

The above regulatory prescriptions are those valid on the date of publication of this sheet. However, given the possible evolution of transport regulations for hazardous materials and in the event of the MSDS in your possession dating back more than 12 months, it is advisable to check their validity with your sales office.

SECTION 15. REGULATORY INFORMATION

This product does not need to be labeled in accordance with EC Directives or respective national laws

Handle in accordance with relevant British regulation: control of substance Hazardous to Health Regulations Environmental Hygiene Guidance: EH40 Workplace Exposure Limits (revised annually)

Labeling: EC regulations:

Further regulations

United Kingdom:

Korea regulations: Industrial safety and hygiene regulation: No Hazardous material control regulation: No Fire prevention regulation: No




Date: 03 / 18 / 2015 Version: 4

Page: 8/88



(704) 276-7100 Fax (704) 276-7101

Ribes Nigrum Fruit Extract: 271-749-0 Exempt Listed: 68606-81-5 Exempt: Ribes Nigrum (Black Currant) Fruit Extract (68606-81-5) Ribes Nigrum (Black Currant) Fruit Extract Ribes Nigrum (Black Currant) Fruit Extract Ribes Nigrum (Black Currant) Fruit Extract

Other regulations:

EINECS inventory status: TSCA inventory status: AICS inventory status: Canadian (CEPA DSL) inventory status: Japan (MITI list): Korea: China inventory status: Philippines inventory status: Listed: Currant, Ribes nigrum, ext.

*Listed on 2010 INCI Standard Chinese Name Directory

Note: The regulatory information given above only indicates the principal regulations specifically applicable to the products described in this sheet. The user’s attention is drawn to the possible existence of additional provision which complete these regulations. Please refer to all applicable international, national and local regulations and provisions

For specific uses, food industry, ask the manufacturer for more information.

01/23/2015

03/18/2015

SECTION 16. OTHER INFORMATION

Prohibited uses:

Last Revision Date:

Preparation Date:

MSDS summary of changes - Added Precautionary Statements - Section 2 (Hazards Identification)- Added Mutagenicity Data – Section 11 (Toxicological Information) & UpdatedTransport Information – Section 14 (Transport Information)

- Added Acute Toxicity Data – Section 11 (Toxicological Information)

The information given is based on our knowledge of this product, at the time of publication in good faith. The attention of the user is drawn to the possible risks incurred by using the product for any other purpose other than which it was intended. This is not in any way excuse the user from knowing and applying all the regulations governing their activity. It is sole responsibility of the user to take all precautions required in handling the product. The purpose of mandatory regulation mentioned is to help the user to fulfill his obligations regarding the use of products. This information is not exhaustive, this is not exonerate the user from ensuring that legal obligations other than those mentioned, relating to the use and storage.


Version#5/03-11-14

PhytoCide Black Currant Powder Manufacturing Flow Chart


Arrival of Materials Arrival of Materials

Tests for Acceptance

Aqueous Extraction of Black Currant (Ribes nigrum) Fruit at a Specific pH and Temperature for

a determined Duration

Make batch adjustments if Needed (Refiltration), if refiltered, material is

also re-spray dried

Initial QC

Pack Material

Sample for Micro

Fail

Pass

Passes Micro

Pass

Fail

Filtration to Remove Undesired Plant Matter

Spray Dry

Ship to Customer

Pass

Final QC

Fail




Code: M16001 INCI Name: Ribes Nigrum (Black Currant) Fruit Extract INCI Status: Approved CAS#: 68606-81-5 EINECS # 271-749-0

The following information on regulatory clearances is believed to be accurate and is given in good faith as a guide to a global use of our ingredients in cosmetic applications. No representation or warranty as to its competences or accuracy is made. Information is offered for use in general cosmetic applications and may vary in particular applications. Users are responsible for determining the suitability of these products for their own particular use. All regulatory decisions should be made on the advice of your regulatory group or legal counsel.

Country / Regulatory Bodyy Status of Product

EU (REACH) Compliant

USA (TSCA) Exempt

Australia (AICS) Compliant

Japan (METI) Compliant

Canada (DSL) Compliant

China (IECSC) Compliant

Brazil (ANVISA) Compliant

Korea (KECI) Compliant

Philippines (PICCS) Compliant

PhytoCide Black Currant Powder Certificate of Compliance





Attention must be paid to the use of PhytoCide Black Currant Powder in the equivalent of OTC formulations (eg. quasi-drugs in Japan, or therapeutic goods in Australia). Some countries maintain restricted inventories of raw materials that can be used in those applications so more detailed guidance may be required.

PhytoCide Black Currant Powder and its components and impurities are in compliance with the rules governing cosmetic products in the European Union (Directive 76/768/ECC & Regulation No. 1223/2009). The recommended use levels for PhytoCide Black Currant Powder is 1.00 – 3.00%.

PhytoCide Black Currant Powder is in compliance with the standardized set of rules developed and approved by the NPA (Natural Products Association).

PhytoCide Black Currant Powder is considered a non-hazardous material. All significant toxicological routes of absorption have been considered as well as the systemic effects and margin of safety (MoS) based on a no observed adverse effects level (NOAEL). Due to the restriction placed on animal testing of cosmetic raw materials, and Active Micro Technologies, LLC’s internal non-animal testing policy, this product was not tested for NOAEL.

PhytoCide Black Currant Powder was tested using in vitro dermal and ocular irritation models. This product was found to be non-irritating in both models.

To our knowledge the above material is free of CMR (*) substances, as defined according to Regulation (EC) No 1272/2008 and Cosmetic Regulation (EC) No 1223/2009 as amended.

(*) Carcinogenic, Mutagenic, toxic for Reproduction

Active Micro Technologies, LLC certifies that to the best of our knowledge our product does not contain any material listed on California Proposition 65.

Active Micro Technologies, LLC certifies that PhytoCide Black Currant Powder does not contain any materials prohibited by Halal laws.

PhytoCide Black Currant Powder is REACH Compliant and free of the following: • Formaldehyde or formaldehyde donors • Parabens• Glycol ethers • Paraffin/petroleum products• Gluten • Phthalates• Lactose • Polyethylene glycol (PEG)• Nanoparticles • Residual solvents• Nitrosamines • Sulfates• Palm oil/palm kernel oil (or derivatives) • Volatile organic compounds

ECOCERT GREENLIFE S.A.S. - Capital 50 000 € – Lieudit Lamothe Ouest – 32600 L’Isle Jourdain - France Tél. + 33 (0)5 62 07 51 09 - Fax : +33 (0)5 62 07 74 96 – www.ecocert.com

TVA Intracommunautaire n° FR 55 509 534 095 CREDIT MUTUEL 2200 20246201 29 - SIREN 509 534 095 RCS AUCH - APE 7120B

ACTIVE MICRO TECHNOLOGIES LLC 4757

VERIFICATION OF THE RAW MATERIALS CONFORMITY TO THE ECOCERT AND COSMOS COSMETIC STANDARDS

Attestation n° :Company:

THIS DOCUMENT IS NOT AN ORGANIC CERTIFICATE

F363(COS)v05en

Page 1 on 4

The present document is only valid for ECOCERT until official COSMOS publication of the raw materials on the website: http://www.cosmos-standard-rm.org/

The conformity (conf.) is established according to the requirements related to the raw materials contained in the applicable standard(s).

*reference related to the appendices II and/or V of the Cosmos standard.

AMTicide Coconut (M14003)

INCI:

Comments:

Function:

Lactobacillus (and) Cocos Nucifera (Coconut) Fruit Extract

Skin conditioning, Hair conditioning

PPAI : 0 % CPAI : 100 % Petrochemical moiety : 0 %

Conf. ECOCERT:

Conf. COSMOS:

YES

YES

0100

0 % synthetic

% of physically processed vegetal ingredients)% of natural origin (

0Non natural ingredient : %

Leucidal Advanced - Aloe (M15015)

INCI:

Comments:

Function:

Water (and) Leuconostoc/Aloe Barbadensis Leaf/Sorbus Aucuparia Fruit Ferment Filtrate

Moisturizing, Skin conditioning, Antimicrobial


Conf. ECOCERT:

Conf. COSMOS:

YES

YES

0100

0 % synthetic



Drawn up in l'Isle Jourdain, valid from

31/12/2015 until

01/01/2015 Matthieu Bouffartigue

Raw Materials Service Manager

WARNING:The approval of the raw material(s) listed above is PERSONAL to the beneficiary named herein, and the BUYERS of the raw material(s) ARE IN NO EVENT AUTHORIZED TO MAKE REFERENCE TO THE APPROVAL BY ECOCERT GREENLIFE OR TO USE AN ECOCERT LOGO, whether in its communication or on the packaging or labeling of the raw material(s) or of a finished cosmetic product.

The present document belongs to ECOCERT Greenlife SAS. It must be erased on ECOCERT request.



Page 2 on 4




Leucidal Advanced - Rowan (M15018)

INCI:

Comments:

Function:

Water (and) Leuconostoc/Sorbus Aucuparia Fruit Ferment Filtrate

Emollient, Skin conditioning, Antimicrobial


Conf. ECOCERT:

Conf. COSMOS:

YES

YES

0100

0 % synthetic



Leucidal Liquid (M15008)

INCI:

Comments:

Function:

Leuconostoc/Radish Root Ferment Filtrate



Conf. ECOCERT:

Conf. COSMOS:

YES

YES

0100

0 % synthetic



Leucidal Liquid PT (M15021)

INCI:

Comments:

Function:

Lactobacillus Ferment

Skin conditioning, Antimicrobial


Conf. ECOCERT:

Conf. COSMOS:

YES

YES

0100

0 % synthetic




31/12/2015 until







Page 3 on 4




Leucidal Liquid SF (M15019)

INCI:

Comments:

Function:

Lactobacillus Ferment



Conf. ECOCERT:

Conf. COSMOS:

YES

YES

0100

0 % synthetic



Leucidal Liquid SF (M15019CHI)

INCI:

Comments:

Function:

Leuconostoc/Radish Root Ferment Filtrate



Conf. ECOCERT:

Conf. COSMOS:

YES

YES

0100

0 % synthetic



PhytoCide Aspen Bark Extract Powder (M16002)

INCI:

Comments:

Function:

Populus Tremuloides Bark Extract



Conf. ECOCERT:

Conf. COSMOS:

YES

YES

100100

0 % synthetic




31/12/2015 until







Page 4 on 4




PhytoCide Black Currant Powder (M16001)

INCI:

Comments:

Function:

Ribes Nigrum (Black Currant) Fruit Extract

Soothing, Skin conditioning, Antimicrobial


Conf. ECOCERT:

Conf. COSMOS:

YES

YES

100100

0 % synthetic



PhytoCide Elderberry OS (M16003)

INCI:

Comments:

Function:

Sambucus Nigra Fruit Extract



Conf. ECOCERT:

Conf. COSMOS:

YES

YES

100100

0 % synthetic




31/12/2015 until





phytocide black currant powder - active micro...

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